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NK cells kill any cells devoid of reduced MHC I molecules on their surface. MHC I molecule is expressed by all nucleated cells except RBCs, sperm cells and others. So how can non-nucleated cells like RBCs escape NK cell killing?
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The answer to your question may lie in the mode of activation of NK cells. NK cells have a two-part trigger: a suppressor and an activator. The suppressor prevents the cell from being activated, while the activator activates the cell to work on a target and destroys the target.
In order for an NK cell to destroy an RBC, both specific activators (ligands) and suppressors (MHC class 1) must be present on the RBC surface. However, since RBCs lack these molecules, they are not recognized as targets by NK cells and are thus spared from destruction.
It's also possible that RBCs further protect themselves by expressing CD47, which is a major recognition molecule of NK cells, or by not having a signal that would fall into the category of DAMP (damage-associated molecular pattern). However, these factors do not change the important point about NK's mode of activation - namely that without specific activators and suppressors present on their surface, RBCs are safe from destruction by NK cells.
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Dear researchers,
Does anybody use anti-CBX7 antibody in western blot? If yes please provide the details of  what company antibody, dilution rates and incubation times.
Thanks for help and suggestions.
-Chakri
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Hello,
I think I could recommend two CBX7 Antibodies from CUSABIO. Both of them have been validated in WB. You can get more details by visiting the following pages:
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I am wondering what happens to the frozen Conditioned-media (CM) that is collected from cancer cells and then incubated for 24 hours to 48 hours? Will the secreted proteins in CM such as Chemokines and cytokines be degraded? In other words, does the incubation affect the CM composition?
Many Thanks,
Fouzia
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In monolayer cell studies, CM is really nutrient depleted medium. This nutrient depletion causes the monolayer cells to spread out more. I.e., become flatter, which causes their nuclei to flatten and changes the chromosomes to change their distribution from 3D to 2D and hence, the number of their nearest neighbors. This halves the number of neighbors for chromosome exchanges and other interactions. The same effect can be produced by diluting fresh media with saline. See the my papers with N.M.S. Reddy, with Peter Mayer, with Joseph Ostashevsky, and with Maria Kapiszewska, all in the 1990s and early 2000s.
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I have read a paper that discussed splenocytes isolation of a group of 5 mice in defined intervals and followed up by 28 days. I wonder how it is possible that you isolate splenocytes on different days separately and keep mice alive for the next isolation. Does it have a different protocol? or small surgery? Or maybe they use another method? it is not clear to me how it is possible in a such small group of mice.
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Hello
l don't know how researchers get splenectomy from the mice where is alive
May be doing surgery but l am getting splenocyte from mice after euthanized means removal of spleen in biological safety cabinet to make splenectomy in tissue culture raw
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I was wondering if anyone has any experience using alternatives for FCS in their HL60 cell culture (or similar cells), I've read that they can sometimes be cultured serum free. However long term I was wondering if there were any alternatives.
I'm happy to do a side by side comparison myself, but any starting input would be great.
Thanks for any help
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Fcs alternatives for HL 60 neutrophil like cell line is FPS (slightly different).
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Hi,
I want to make the IsoFlow Sheath liquide in our lab by following the chemical composition given by the company ( mentioned down below) , i want to know if any of you tried this before and if it works without any risk during the aquisition process. and i would like to know also if there are any tips or precautions to take when preparing the solution.
Chemical composition for 10L :
Sodium Chloride......................7.93 g/L
Disodium EDTA........................0.38 g/L
Potassium Chloride.................0.40 g/L
Monosodium Phosphate.........0.19 g/L
Disodium Phosphate..............1.95 g/L
Sodium Fluoride......................0.30 g/L
Kind regards.
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IsoFlow in Beckman coulter or sheath fluid in BD machine are the isotonic buffers solutions which may be replaced by Autoclaved and filter sterilized PBS.
You should be extra cautious while using in-house sheath buffer as any particle or growth may block your tubes in flowcytometer.
We have used Autoclaved and filter sterilized PBS in BD instrument and it never caused any issue.
Once you use any in-house buffer, the parent company will consider it void of warranty. So, never let the company people know that your are not using their isoflow.
Good luck,
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Hi,
I'm about to start a proliferation test for lymphocytes based on CFSE dye, but i don't have much knowledge in cell culture field. I would like to have some help from an expert on this field who has a valide protocol for that functional test.
Best regards.
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Please see my CFSE proliferation protocol attached and two papers for the results. The outline is for B cells, but it's easily adaptable to any other lymphocyte population.
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We have recently seen a case of large diffuse B-cell lymphoma of probable folicular origin (CD10+ partial expressión) with a well defined CD103 expression by B-cells. The references in the literature are scanty and unclear.
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Jordi Juncà We have just analyzed the blood of a patient with previous diagnosis of follicular lymphoma in lymph node that returned to the hospital with adenopatias and abnormal lymphocytes in blood. We performed an immunophenotyping of the blood and found 12% of medium sized cells with characteristics of follicular lymphoma: CD19 weak, CD10++, BCL-2++, CD5 neg, CD43 neg and IgM neg. However, these cells showed some positivity for post-germinal markers such as CD27, CD11c and CD39. In addition, 50% of the abnormal population showed expression of CD103 (without expression of CD25, CD123 or LAIR-1). It is the first time I see CD103 expression in a follicular lymphoma. On the other hand, I´ve already seen cases of hairy cell leukemia (very classic) with CD10 expression.
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Usually if immune response to molecule A isn't elicited, then another known immunogenic molecule B is conjugated to A and delivered into the body.
The response is that the immune system elicits antibodies against molecule A, molecule B and molecule A-B conjugate.
Then in that case, do live vector vaccine delivery systems like Lactococcus/E.coli also get an immune response against them?
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I think yes.
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Hi,
I am staining a human blood with CD19, CD3,CD14 and CD16. My gating strategy in FlowJo goes like this:
- First, I gate a general population SSC-A VS FSC-A => Then gate for single cells, => after that I try to identify Lymphocytes, Monocytes, and Neutrophils.
- Then, I look for CD16 VS CD19 in Lymphocytes gate to identify the B cells, NK cells and T cells by gating first on CD14- then CD16 VS CD19.
- In the monocytes gate, I try to distinguish the monocytes sections( classic, non-classic and intermediate monocytes) by using CD16 VS CD14 markers.
- Lastly, in the Neutrophils, I gate on CD14 -- and CD19-- to identify CD16.
I am not sure about this gating strategy I found it a bit complicated and I am wondering if there is any sample way to identify the Lymphocytes, Monocytes, and Neutrophils in a human blood using this 4 markers!
Also, my problem is that I find it some time difficult to identify the monocytes and to distinguish the monocytes sections( classic, non-classic and intermediate monocytes).
Do you have any advice and tips to improve the staining and the gating strategy for better identifying the monocytes and Lymphocytes?
Thank you for your time and help.
Arwa
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The CD16± NK cells is problematic. Cells often have small populations that change shape, eg neutrophils found in monocyte or lymphocte size area, meaning your CD16± gate may not be reliable for NK(CD56, is most ideal) To clear up the B cell and monocytes from the rest, good to add HLADR.
Regarding the monocyte subsets, this is usually influenced by time it takes to process blood and the anticoagulation type.
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Are macrophages that are elicited to peritoneal cavity after the injection of agents (thioglycolate, gelatin, peptone) inflammatory? Do such elicitating agents have the same effect on their biology?
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Hey Emilia.
We use peritoneal macrophages as standard ex vivo model in nearly every experiment (besides BMDM and MEF sometimes) for a long time but I recommend to not use thioglycollate or any kind of elicited PMs.
I know everybody is using it to get more macrophages (because of limited mouse numbers) but my colleauge and I had to compare normal peritoneal macrophages and those attracted via thioglycollate injection during our PhD works. Thioglycollate-elicted macrophages respond in nearly every parameter we investigated (bacterial killing, ROS production, cytokine prodcution, phagocytosis capacity) much worse than normal peritoneal macrophages.
They are more or less powered out. They used their whole pro-inflammatory potential on the thioglycllate and ate themselves (so to speak) to laziness.
They phagocytosed less bacteria, they prodcued nearly no ROS (Nox2-dervied phagosomal or cytosolic ROS from mitochondria) and they secreted nearly no cytokines after infection (all compared to PMs without thioglyllate).
Moreover, the injection of thioglycollate is also a sterile inflammation, so other cells will be recruited from the blood stream (monocytes, neutrophils).
Again, I know everybody is using this technique, but this hardly represents an ex vivo peritoneal macrophage.
Of course, in dependency of the research topic, not only PMs but different ex vivo macrophages should be picked for the experiments (e.g. Kupffer cells for liver, microglia for brain and so on).
All the best and stay healthy,
Marc
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Antigens are processed by specialized macrophages to produce complex protein-RNA complexes that eventually produce iRNA. When this iRNA is introduced to primary B cells that have never seen the antigen, they produce specific antibodies to that antigen. Th macrophage produced RNA is incorporated into the the genome of these B cells by reverse transcriptase that now become "memory cells" capable of a secondary response when confronted with the antigen they had never come into contact before. Reverse ttranslation in the macrophage is the best explanation for the production of such specific iRNA.
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Yunus Shukor Thanks.
Indeed between a choreographed dance of hypermutations and recombination, gene splicing and translocation of gene segments to generate an enormous variety of VxDxJxC expressions for an antibody or the existence of a reverse translatase that would process the exposed epitope of an antigen and encode an RNA segment for the hyper variable segment of an antibody, nature and Occam‘s Razor I feel would pick the latter.
Thanks you M. Y. Shukor.
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Dear,
We analyzed a patient of 11 months for a possible Primitive Immunodeficiency Disease, we made an immunophenotyping and the outcame was :
Lymphopenia of LT-CD4+
Lymphocytosis of LB-CD19+
Lymphopenia of CD45-RA
Lymphocytosis of CD45-RO.
I searched for a possible PID but i didnt found anyone that match with this profil. I'll appreciate your precious help.
Best regards.
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What is "Primitive Immunodeficiency"? I know only primary immunodefiiciency.
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The neutralisation of the coronavirus has been much discussed in recent weeks, including antiviral medication used for HIV, HCV and anti malarial drugs with variable outcomes (potent antiviral drugs, such as remdesivir, chloroquine, or lopinavir/ritonavir) also convalescent plasma and IgG. However, inflammatory mediators appear to impact the progression of disease in COVID 19 patients.
Viral infection require precise regulation of the innate immunity by inflammatory immune mechanisms but over-activation of these processes can cause immunopathology with further complications to infected patients. Some significant clinical feature or patients with coronaviruses include, dyspnea, hypoxemia, and acute respiratory distress, lymphopenia, and cytokine release syndrome. This suggests that homeostasis of the immune system could play an important role in the development of COVID-19 pneumonia. Some plasma cytokines and chemokines are increased in COVID 19 patients, including IL-1,2,4,7,10,12,13,17, GCSF, MCSF, IP10, MCP-1, MIP-1, hepatocyte growth factor, IFN-γ and TNF-α.
The protective barriers of mast cells of the submucosa in the respiratory tract are activated by the virus and release histamine and protease and later activate IL-1 and IL-33. Could IL-1 receptor antagonists be helpful?
Histamine, as well as affecting vascular and bronchial responses, is increasingly identified with modulation of immune responses, including a variety of lymphocytes, such as T cells. Could antihistamines have beneficial effects on immune dysregulation and tissue remodelling during COVID 19 infection?
Virus particles invade the respiratory mucosa firstly and infect other cells, triggering a series of immune responses and over-activation of lymphocytes by apoptosis or necrosis of infected cells and the production of a cytokine storm causing a systemic T cell response in the patient, which may be associated with the critical condition of COVID-19 patients. COVID 19 attaches to pulmonary host cells by ACE2 then fuses to the membrane and releasing viral RNA. Lower levels of granulocytes are observed in the severe group than the mildly infected.
The development of inflammatory complications may be associated with the genetic individuality of a patient’s innate immune responses, resulting in different phenotypes. Considering the balance of IL-10/IL-12 expression influences the Th1/Th2 responses and imbalance in airway mucosa plays an important role in immune responses to viral infections and asthma development, IL-10 drives a humoral response and IL-12 drives a cytotoxic T cell response. Whereas Th2 responses are linked to the development of atopy, Th1 differentiation is often associated with the pathology of certain autoimmune processes. Patients with asthma viral infections tend to promote a Th2 response and increased eosinophilia exacerbates symptoms of the disease leading to breathing difficulties. Patients with chronic airway inflammatory diseases have impaired or reduced ability to promote Th1 cytotoxic responses to neutralise the virus. Could this be an implication for IL-12 therapy for anti-viral responses in patients not able to clear COVID 19?
In the severe group, CD4+ cells with lower IFN-γ and TNF-α and levels of granzyme B and perforin in CD8+ T cells were higher in the severe group than in the mild group. Could IFNγ as an antiviral therapy, despite its rather unpleasant side effects?
Further, Zinc supplementation showed benefits, shortening the duration of oxygen desaturation, tachypnea, and clinical symptoms in children with pneumonia, showing a Th1 response with the increase of IFNγ and IL-2 cytokines.
Chloroquine also seems to act as a zinc ionophore, thereby allowing extra cellular zinc to enter inside the cell and inhibit viral RNA dependant RNA polymerase.
Please contribute to this discussion.
Suggestions for anti-inflammatory considerations
  1. Antihistamines administered early in infection may reduce excessive cytokine proinflammatory storms.
  2. Zinc supplementation of population
  3. IFN-γ
  4. Introducing anti-inflammatory cytokines and/or monotherapy blocking IL-1 cytokine or receptor, inhibiting IL-1 may inhibit the inflammation.
  5. IL-4,6,10,11 and 13 are anti-inflammatory cytokines
  6. IL-1 receptor antagonists
  7. Chloroquine, the antimalarial drug that inhibits lymphocyte proliferation. As well as anti-viral activity. US have approved this therapy.
References
  • Marone G, Granata F, Spadaro G, Genovese A, Triggiani M. The histamine-cytokine network in allergic inflammation. J Allergy Clin Immunol. 2003;112(4 Suppl):S83–S88. doi:10.1016/s0091-6749(03)01881-5
  • Yan-Rong Guo, Qing-Dong Cao, Zhong-Si Hong, Yuan-Yang Tan, Shou-Deng Chen, Hong-Jun Jin, Kai-Sen Tan, De-Yun Wang, and Yan Yan The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak – an update on the status. Mil Med Res. 2020; 7: 11.
  • Zheng, H., Zhang, M., Yang, C. et al. Elevated exhaustion levels and reduced functional diversity of T cells in peripheral blood may predict severe progression in COVID-19 patients. Cell Mol Immunol (2020). https://doi.org/10.1038
  • Front. Pediatr., 14 November 2019 | https://doi.org/10.3389/fped.2019.00431). /s41423-020-0401-3).
  • J A Carr, J Rogerson, M J Mulqueen, N A Roberts, and R F Booth. Interleukin-12 exhibits potent antiviral activity in experimental herpesvirus infections. J Virol. 1997 Oct; 71(10): 7799–7803.
  • Jorge Alberto Acevedo-Murillo, Miguel Leonardo García León, Verónica Firo-Reyes, Jorge Luis Santiago-Cordova, Alejandra Pamela Gonzalez-Rodriguez2 and Rosa María Wong-Chew, Zinc Supplementation Promotes a Th1 Response and Improves Clinical Symptoms in Fewer Hours in Children With Pneumonia Younger Than 5 Years Old. A Randomized Controlled Clinical Trial. Front. Pediatr., 14 November 2019 | https://doi.org/10.3389/fped.2019.00431
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Thank you so much, Ruediger, for your contribution. I made a few notes on mast cell activation, particularly IgG involvement as over the last year mast cells have been a particular interest for me and am preparing a paper on the subject.
Mast cells
They originate from CD38+, being granulocytes derived from myeloid stem cells. MCs are also associated with neuro-immune systems. As innate immune cells, MCs are phenotypes that are tuned by cytokines and other micro-environmental stimuli. They also play a role in transplantation immunology but this will not be further discussed in this review.
As members of the innate immune system, MCs are located in places like skin, lung, and intestinal mucosa, which are in close contact to the outside world and they have an immediate activated response to parasite infections and antigens involved in allergies. In the case of parasites and allergens, these long-lived MCs defend against parasites maintaining an immune protection on the physical barriers in the body and are activated by the antigens. The response is mediated by a cross linking of FceR1 by immunoglobulin E (IgE), which results in the degranulation of the MCs. This particular linkage is used in MC detection by applying high infinity IgE. Staining the MC granules with toluene blue also characterises the cells.
There are a wide variety of receptors expressed on the surface of MCs that enable them to be activated by several different ligands, such as, endogenous cytokines, IgE, TLR ligands and IgG immune complexes. The inflamed tissue contains many of these ligands. A number of mediators including chymase, tryptase, histamine and other cytokines and chemokines are released, dependent on the route of activation.
An in vivo experiment with rats found that IL-3 wa involved in the recruitment of MCs and there were differential effects, dependant on the target tissue and time of exposure to the chemoattractant (de Cássia Campos MR 2014). Other mediators that stimulate MCs and trigger degranulation, proliferation and release of mediators include, IgG, complement components, TLR ligands, neuropeptides, cytokines, chemokines as well as other inflammatory products. Migration and differentiation are also stimulated by these inflammatory components. Thus the true versatility of MCs is recognised through them responding to a wide repertoire of different stimuli and not just IgE involvement (Yu Y 2016).
The MRGPRX2 MC receptor is important in the activation of MCs by peptide stimuli with abundant positive charges and aromatic/aliphatic amino acids (Lu L 2017).
The activated MC response results in the release of a broad spectrum of proinflammatory mediators, proteolytic enzymes and chemotactic factors that attract other immune cells. Proteolytic induce rapid inflammation and tissue remodelling.
MCs also are important in wound healing releasing factors and promote the recruitment of leukocytes, platelet activation and fibrogenic processes. Although the pathogenesis of RA is not fully understood, joint destruction possibly occurs due to the recruitment of neutrophils and monocytes that facilitate the damage of joint related cartilage when they activate osteoclasts. Histamine is a mediator that is involved in the activation of osteoclasts.
Clearly, in the case of allergies this immune response is detrimental and can cause asthma, even promote anaphylaxis. The MC is rich in granules of histamine and heparin, all-important in the fenestration of epithelium to enable other immune cells, such as, lymphocytes to migrate to the source of supposed infection. Other mediators of immune response from MC granules are leukotrienes causing shortness of breath, prostaglandins, tryptase, interleukins, heparin and TNF-alpha (Jennings S 2014), (Afrin LB 2013), (Valent P 2012), (Theoharides TC 2015).
MCs promote anti-inflammatory mediators as well as proinflammatory processes. They can act as antigen presenting cells and express a large array of co-stimulatory molecules. MCs are able to tolerate the introduction of some antigens without eliciting an inflammatory immune response in certain sites of the human body, described as having immune privilege to tissues with T-regulatory cells and are essential elements in fibrotic conditions.
In allergic responses, MCs are a potential source of chemokines and cytokines, important in inflammation. As well as IgE activation, MCs can be activated by Toll-like receptors and/or IL-1, which can be inhibited by IL-37 whereas IL-36 is a powerful proinflammatory cytokine (Gallenga CE 2019). IL-1 activates the release of inflammatory chemical mediators from MCs.
There are many known triggers for MC activation in addition to Infections (viral, bacterial or fungal)-
· Stress: emotional, physical, including pain
· Stress: environmental (i.e., weather changes, pollution, pollen, pet dander, etc.)
· Food or beverages, including alcohol
· · Heat, cold or sudden temperature changes
· Exercise
· Fatigue
· Drugs (opioids, NSAIDs, antibiotics and some local anaesthetics) and contrast dyes
· Natural odours, chemical odours, perfumes and scents
· Venoms (bee, wasp, mixed vespids, spiders, fire ants, jelly fish, snakes, biting insects, such as flies, mosquitos and fleas, etc.)
· Mechanical irritation, friction, vibration
· Sun/sunlight
Typical symptoms experienced during mast cell activation in allergy are flushing; itching, diarrhoea and hypotension are all mediated by histamine. Leukotrienes cause shortness of breath and prostaglandins are responsible for pain, brain fog, cramping and also flushing. In addition, MCs induce cytokines that cause fatigue, weight loss and enlarged lymph nodes. MCs are also implicated in autoimmune pathology as well..
Perhaps patient histamine levels may be used as an early indication of COVID 19 disease severity or outcome by measuring the COVID 19 patient levels of histamine in serum and urine to assess the possible degree of mast cell involvement in the cytokine storm.
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I have used the following markers in a flow cytometry panel to assess mouse samples (7 days old mice and 30 days old):
F4/80
CD45
CD11b
I isolated peritoneal macrophages and bone marrow monocytes. In the side- and foward- scattern are in both tissues two clouds (macrophages? monocytes - mature/immature? granulocytes? lymphocytes?).
I would be happy about some advice as to what cells I can identify with these markers and a gating strategy!
Thank you,
Felix
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concerning your question:
In brief, for the gating strategy, please see attached:
Macrophages are often defined as CD11b+/F4/80high, whereas monocytes are defined as CD11b+/F4/80dim, which is a bit arbitrary, without additional markers.
The supplementary figure is from the following paper in PNAS:
Just three general comments to improve macrophage/ monocyte flow cytometry data, as I don't know the details of your experiment.
1. Did you stain for live/ dead cells? This will certainly reduce your background, as antibodies randomly stick to dead cells.
2. Did you block Fc receptors on your cells by possibly using purified Rat Anti-Mouse CD16/CD32 (clone  2.4G2) to reduce your background? Macrophages/ monocytes express different Fc receptors on their cell surface and these will bind your antibodies used for FACS with different specificity, if you do not block them before.
3. Did you use FMO controls to define your gating strategy as laid out in the paper, which is the only way to .
All the best & good luck with your experiment,
Michael
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NK cells or interferon alpha? 
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The immune response that develops in a viral infection is first given by the innate immune response. The virus enters the cell and deposits its genetic material that is recognized by endosomal TLRs (TLR3, 7, 8, 9) or by proteins of intracytoplasmic recognition (MDA-5, RIG-1 and DAI) of foreign DNA and RNA, these proteins recognizing foreign nucleic acid will activate the kinase proteins that will then activate the transcription of IRF (interferon regulation factor, IRF1,3 and 7 ) that enters the cell nucleus and activates the transcription of interferon type I (alpha and beta). This cell will produce type I interferon that comes into contact with neighboring uninfected cells by interferon I and II receptors, getting the cell into an antiviral state. This state is caused by the activation of the transmission pathways of jack-stat signals that induce the expression of genes whose products interfere with the replication of the virus in uninfected cells. We must remember that type I interferon (alpha and beta) increases MCH-I expression, activates NK cells and promotes differentiation of Th virgin lymphocytes to Th1.
I hope the information provided helps you, regards.
Español
La respuesta inmune que se desarrolla en una infección viral se da primeramente por la respuesta inmune innata. El virus ingresa a la célula y deposita su material genético que es reconocido por los TLR endosomicos (TLR3, 7, 8, 9) o por las proteínas de reconocimiento intracitoplasmatico (MDA-5, RIG-1 y DAI) de ADN y ARN extraño, estas proteínas al reconocer ácido nucleico extraño activaran las proteínas cinasas que luego activaran la transcripción de IRF (factor de regulación del interferón, IRF1,3 y 7) que ingresara al núcleo celular y activara la transcripción de interferón de tipo I (alfa y beta). Esta célula producirá interferón de tipo I que entrará en contacto con las células vecinas no infectadas mediante los receptores de interferón I y II, consiguiendo que la célula entre en un estado antivírico. Este estado se da por la activación de las vías de transmisión de señales jack-stat que inducen la expresión de genes cuyos productos interfieren en la replicación del virus en las células no infectadas. Debemos recordar que el interferón de tipo I (alfa y beta) incrementa la expresión de MCH-I, activan las células NK y promueven la diferenciación de los linfocitos Th vírgenes a Th1.
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"The rate of point mutation in the V region is estimated to be about 1 change per 1000 base pairs per cell division, about 1000 times the rate of somatic mutation for non-Ig genes"
Does it mean that in non Ig genes mutation occurs every 1 million bases?
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Somatic hypermutation (SHM) introduces mutations in the variable region of immunoglobulin genes at a rate of approximately 10(-3) mutations per base pair per cell division, which is 10(6)-fold higher than the spontaneous mutation rate in somatic cells. To ensure genomic integrity, SHM needs to be targeted specifically to immunoglobulin genes. The rare mistargeting of SHM can result in mutations and translocations in oncogenes, and is thought to contribute to the development of B-cell malignancies. Despite years of intensive investigation, the mechanism of SHM targeting is still unclear.As per above statement mutations occurs every 1 million bases
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We are working with murine CD8 T cells at the moment and for this sake we isolate CD8 T cells with the Dynabeads untouched mouse t cells kit (thermo fisher). So for several weeks we didn't have any problems and a quite good efficency. But since three weeks we are struggling with the isolation because our cells cannot be stimulated and die after seeding them. We didn't change any steps in the protocol. So for better understanding this is the rough protocol we use:
- Smash the spleen with the end of a 2 ml syringe and filter throgh 40 um Nylon mesh
- suspend and wash the cells in cold PBS + 0,5% BSA + 2mM EDTA
- resuspend the pellet in 1x erylysis buffer for 2 min, fill up with cold PBS + 0,5% BSA + 2mM EDTA and wash
- block the cells with FCS and follow the instrustions from the isolation kit to isolate CD8 cells (we never changed how we did this: incubating cells with antibody mix, washing beads, incubate cells with beads, remove beads, transfer supernatant containing CD8 cells, wash beads)
- resuspend the isolated CD8 cells in media + 10% FCS, add 100 U/ml IL-2, add CD3/CD28 activator beads (Ratio 0,8:1), add 50 uM beta-ME
- seed the cells in a density of 1*10^6/ml in a 24 well plate
After 3 days we normally saw a good proliferation and cells sticking to the beads.
Since three weeks the cells just seem to die. After 3 days the density is no more than 0,2-0,3*10^6/ml. The cells don't stick to the beads and don't look stimulated. Using flowcytometry we sometimes saw a lot of celldebris (as we suspect) in our suspensions even shortly after the isolation.
We double checked every solution we used.
So we used fresh PBS +0,5% BSA +2mM EDTA and we diluted fresh 1x erylysis buffer from our 10x stock solution. Those solutions are being used by most of the people in our lab and no one esle is having any problems like this. We have two different operators doing the isolation. Each of them are preparing two different mice.
So we opened a new bottle of activator beads and saw no difference.
We then came to the conclusion that it might have something to do with the isolation kit. So we skipped the isolation in our protocol and stimulated the splenocytes which seemed to work as far as possible. We then used a different kit for a positive isolation and this worked too, but the efficency is quiet low which is why we would rather stick to the negative isolation.
We ordered another Dynabeads untouched mouse t cells kit with a different lot number which we used today.
We don't now yet if the cells can be stimulated but our flowcytometry analysis looked horrible. Again there was a lott of cell debris and only around 7% lymphocytes (normally around 80%).
So now my question is: does anybody have experience with a problem like this or any kind of suggestion what we could check again?
Thank you.
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Annick Fehrer Hi i read about your struggles, and I'm glad that you have solved the issue! I was, however, intrigued by your last message- do you have any sources where I could read about isoflurane impact on immune cells/ how did you pinpoint this as the problem? Thank you in advcance!
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Can a cellular mediated immunity vaccine inhibit the production of antibody post-infection ?
In other way, if you use a cellular mediated immunity vaccine against an infection naturally triggering an antibody-dependent immunity (protective or not), does the infection produce antibody or the vaccine orientation predominate ?
The first immune orientation is difficult to change (like for allergy, viral infection ..)
Am I right ?
Please support with data or publication
Thank you
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Dear It is possible to stimulate the production of antibodies Post infection.
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I am currently optimising a plate-bound adhesion assay to study the adhesiveness of polyclonal human T-cells to differing concentrations of ICAM-1. 
I start with coating the plate with ICAM-1-Fc. I usually do this overnight, and then, after 3 washes with PBS (at room temp) + 40mM HEPES + 2mM Mg2+, I block the plate with PBS-3%BSA.
I then seed CFSE labeled cells onto the plate. The assay/binding buffer I use contains RPMI + 0.1% BSA + 2mM Mg2+ + 40mM HEPES.
After 20 minutes of incubation at 37C, I gently wash off the cells (x3), and then read the fluorescence on a plate reader.
What I have consistently noticed is that in the blank or 0 ug ml-1 ICAM-1-Fc condition, adhesion is the greatest. I have no idea what these cells are adhering to, but I am wondering if it is the BSA, perhaps? 
So, folks who commonly do plate-bound adhesion assays, do you or do you not block the plate after seeding your ligand? 
The plates I am using are opaque-walled, 96 well plates, polystyrene bottomed. 
Thanks, folks!
A.
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Marta Lopes and Asma Ejaz The last time I ran this assay was in, oh, 2015? :-) But here is what I did:
1) I switched to untreated glass-bottomed plates from iBidi, and I acid-washed them with acid-alcohol (HCl and EtOH, but the concentrations escape me now).
2) Then, I'd coat the wells (or the chamber slide, for imaging purposes) with poly-L-Lysine at 0.1mg per ml: this you can coat at RT for 5-15 mins, wash off with sterile water, and let dry at RT overnight.
3) The saturating conc of recombinant human ICAM-1 was 5 micrograms per mL in my hands. I'd dilute this in PBS-0.1% BSA, and coat the slides/plates at RT overnight or at 37 for 2 hrs.
4) Wash off the ICAM with PBS, and block with PBS-5% BSA for an hour at 37C.
5) 3-4 washes with water.
6) Your cells are now ready to be seeded. Once you plate your cells, I like giving them a quick low speed spin: 30 seconds at the most. Literally, I set the centrifuge at 400xg, pop the plates in, hit start, and hit stop the moment it gets to 300. The centrifugal force gently pushes the cells down onto the surface enabling them to make quick contact with the ICAM-1.
6.1) I prefer to plate the cells in phenol red-free medium with 5% FBS/human AB serum (depending on your cell type). My favourite medium to do this is FluoraBrite. Should you choose to image the cells, the fluorescence is much clearer and sharper in this medium.
7) The plate should now be placed at 37C for 90-120 mins, after which they should be subject to 4 gentle washes with a warm (37C) wash buffer...which is essentially the medium you plated the cells in. I also recommend using wide-bore pipette tips for this and dripping the wash buffer from the side of the plate rather than directly blasting the cells with wash buffer. Shear force can sometimes loosen adhered firmly adhered cells, and we only want to get rid of those who cannotb commit to the ICAM-1. :-)
8) You can either pipette out the wash buffer, or just flick it out of the plate. Literally, pick the plate up, flip it over and with a gentle flick of your wrist, dispose of the wash buffer.
9) As far as a read out, there are few ways: I preferred fluorescence, but I hated CFSE because of how many cells it killed off, and I was working with a pretty rare population from PBMC anyway. Cell permeant dyes like CellTracker Green or Violet are gentler on the cells, so I recommend those.
10) Once you've performed your washes, you can pop the plate in a plate reader capable of reading fluorescence, and the magnitude of fluorescence is directly proportional to how many cells stuck around after washing, and thus can be thought of as a function of adhesion.
Certainly, +ve and -ve controls are vital for these assays with the view of subtracting background. I'd also include an unwashed control, where the cells are allowed to adhere, and are not washed. When read on the plate reader, the values this condition give you can be thought of as 100% or max adhesion, and you can normalise your treatment conditions as %of max to get a cleaner/crisper graph to wow everyone at lab meeting. :-)
All the best! I am sorry this is so scattered, but it's been a while! Feel free to reach out if there're questions, and I'll do my best to solve them.
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I work on evaluate the cytotoxicity of herbal extract on primary human monocyte-derived macrophages (MDMs). When I'm doing MTT assay on 96-well plate, I scrape MDMs from cell culture flask and seed in 96-well plate with density 50,000 cells/well. However, after overnight incubation most of MDMs are not attach to the surface and lost during washing step in MTT protocol. So, I think it might come from the high density of the cells and there is no sufficient surface area for MDMs to attach(?) or the cells just died after cell scraping (?) Could anyone please suggest me on the optimal density of primary MDMs in cell culture plate or other tips for handling with MDMs culture would be greatly appreciated.
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1. do not subculture your cells
2. it is generally considered that there is 9-10% of monocytes are present in healthy volunteer buffy coat.
3. after counting of cells i have followed the below numbers:
in 24 well 3 million PBMC in 2.5mL ( approx 1 million per mL) ( final monocyte count is 0.3 million( you can count a representative well)
in 96 well 1 million PBMC in 250uL ( final count is 0.1 million)
hope this helps you
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To standardize immunoassays.
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Hi, related to this question we recently obtained a pool of 129 hiv-1 Gag peptides (15 Mer) from the NIH. They sent us individual peptides lyofilized, and although they provided the amount (1mg each) and the mollecular weight, but were not very clear regarding the volumes we should use to resuspend them or the range of concentration to build our stock and combine the peptides. We are hesitating on whether to use the same volume (we obtained the same mass from each and they all have very similar length and Molecular weight) and directly combining them, or to calculate for each of them a given Molarity at which volume we should resuspend the peptides. Do any of you happen to have a protocol for this or would be so kind as to give us few tips based on your experience? Any information will be greatly appreciated! Best regards, Enrique
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I need to grow mouse Beta-TC-6 in the lab, I searched for the medium protocol but did not find any published data?
Do anyone have an experience culturing them?
what is the protocol of the medium?
should I add 2-Mercaptoethanol to the growth medium?
Thanks
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The medium:
DMEM Low Glucose + Glutamax
- 10% Heat Inactivated FBS
- 1% P/S/A
- 9,725ml of 1M D-glucose
I started 3 years ago and i have never changed the medium composition (even if ATCC did it).
Almost all my cells are positive for Nkx6.1 and more than half are positive for INS and C-peptide.
Really easy...except one thing, these cells grow in cluster.
Do not wait for confluent culture.
In this case, they die (or at least they detach from the plastic).
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I will be treating heparinized whole blood with a small molecule for various timepoints and then extracting RNA for qPCR.
Do people recommend incubations in tubes or plates/dishes?  5% CO2/37C or only 37C?  I am set on the RNA extraction part, but fairly inexperienced with the prior steps, so any hints or tips would be well appreciated.
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following
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Is it possible to used the rabbit polyclonal antibodies as an antidote for human and is that legal to be injected to human?
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Forget the legality of it all...Never a good idea to inject animal protein of any sort into humans. You will induce an anti-rabbit antibody response in the person. And if the person gets another dose (or somehow gets exposed to rabbit antibodies) after a memory response is induced... it'll cause a type III hypersensitivity.
There are very limited cases where this is okay... the most obvious one is anti-venom (which is horse serum/polyclonal antibodies against venom, usually snake).
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I want to stain intracellular cytokines and exhaustion markers (e.g. PD-1/CTLA-4) in mouse LN/spleen samples. I'm wondering whether ex vivo Ag restimulation or anti-CD3/28 stimulation affect expression levels of exhaustion markers ?
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you should consider that the expression level of CD28 and IL-7R on exhausted T cell are declined. if you stimulate these receptors, you may no see the result as much as is expected.
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Hi all, I try to activate CD4-T cells isolated from PBMC using Dynabeads® Human T-Activator CD3/CD28 (Invitrogen) with cell/beads ratio 1:1 (1 million cells/well in 6-well plate). After 48 hours incubation, I collect cells and measure cell cycle by flow. It looks that my experiment don't work 'cause 99% cell still stay in G1 phase, like untreated CD4-T cells. Is there any trick in it? Any suggestion will be deeply appreciated! 
George
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I also failed to perform this experiment. I used positive selection T cell. I am afraid that that is the big problem for T cell activation. Can you share me more experiences?
Thank you so much.
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Currently I am working on AAV triple transfection. According to the paper Lock,etal, human gene therapy, 2010. AAV virus can release from cells into media. We do not have TFF as what they used in their core lab. Any other replacement method? Thanks a lot!
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Usually if you have good enough transfection of 293 (https://altogen.com/product/293-transfection-reagent-emb-kidney-crl-1573/) you can get the virus to be filtered with simpler methods. I would look at incubation protocols and Darrick's answer for more information.
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About flowcytometry.
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this is the beauty of flowcytometry dear.
By flowcytometry you can actually analyse the co-expression of multiple cell markers on a single cell. number of markers to be analysed depends on the filters available in your flowcytometer, that may vary from 2 color to 17 colors.
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We have been trying to isolate monocytes from mouse spleens using StemCell's monocyte isolation kit. However, our yields have been terrible (e.g. 200*10^6 splenocytes --> 1*10^6 macs). I heard that splenic macrophages digest RBC's and will stick to the magnetic collumn due to the ingested iron. Further, we would like all CD11b positive cells in the spleen, not just the monocytes. The reason for using negative selection as opposed to positive would be to minimize the effects of bound metallic antibodies.
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CD11b positive cells in the spleen are not very abundant and when I have tried them yield is very poor. I switched to the the bone marrow system. I plate the cells in the complete media and after 7 days you should have a very enriched and purified population.
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Are there specific encoded proteins that are responsible for a majority of the adipogenic effects of Ad-36? If you were to clone a specific subset of Ad-36 encoded products into a lenti and infect cells, which would you use?
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Hey Robert, did you ever find an answer to this question?
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I want to know if there is any specific test for evaluating cross-presentation (in terms of protein or gene expression or something else) in dendritic cells isolated from human blood. If there are some methods, which one is the best?
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So many thanks for your valuable recommendations. They did help alot.
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I need human neutrophils for performing all of my experiments. It is very difficult for me to isolate neutrophils each time and perform experiments. Is their any neutrophil cell line available whose data will be accepted in research paper?
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Collins lab in UC Davis have performed gene expression studies of two known well lines that can be differentiated into neutrophil-like cells and the expression pattern suggest that PLB985 and HL60 may be very very similar in their origin , if not identical. Either cell line would work well for certain applications. These cell respond to chemotactic stimuli and will migrate comparable to primary neutrophil. However, their ability to form NETS and production of certain cytokines vary vastly. So choose your poison accordingly.
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I am trying to look at expression of mature IL-1 beta and caspase-1 activation at protein level. I am not very sure for how long I need to treat my cells with Tunicamycin or Thapsigargin to see an increase in expression of desired proteins. Any suggestion for the conditions required will be highly appreciated.
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 Thanks Dr. Ponnuraj.
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Which techniques are most appropriate for quantifying an antibody in a sample?
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Enzyme-linked immunosorbent assay (ELISA) based methods are the most efficient in quantifying the level of antibody in a given sample.
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Thanks in advance for your replies.
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Dear Bruce
Thank you for your kindly reply.
Best regard
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My supervisor has asked me to find a protocol for freezing and thawing naive T cells, with details regarding the % recovery of the T cells once thawed, but I'm struggling to find any information online.
Has anyone got an experience with freezing and thawing naive T cells?
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To add to what Zaid said, I typically use benzonase in my thawing buffer to decrease the stickiness caused from DNA being spilled by lysing cells.  I do my spins at 200g for 10 minutes, room temp, brake <3.
BTW when you search for protocols you should just look for general PBMC freeze/thaw protocols.  There are thousands out there. 
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As part of an effort to determine whether a certain proliferative response of virtual/innate memory T cells (CD44hi) I tried to knock-out the TCRa of these primary T cells and to culture them with my stimulation of question or with what I thought to be a TCR-independent stimulation, PMA/Ionomycin. I wanted to then, after 96 hours of culture, check for the ratios of TCRb-positive vs. TCRb-negative T cells in both cultures, assuming the PMA/Ionomycin stimulation will, on one hand, keep those primary T cells alive and well during those 96 hours, but on the other hand will get them to proliferate in a completely TCR-independent fashion. This does not seem to work, however, and more than 90% of the cells in that culture die (should I try it without ionomycin maybe?).
My questions are, therefore:
1) What would be a proliferation-inducing stimulus which I can use for T cells and which is practically/completely TCR-independent? As these are innate/virtual memory CD8+ T cells, maybe I should use IL-15/IL-7/both?
2) Do you have any suggestion as to how I shall examine the TCR-specificity of a reaction of innate/virtual memory CD8+ T cells without manipulating MHC presentation (KO of b2m from the immunogenic cells gave ambivalent results, which is the reason I wanted to check the other side of that coin, the TCR-dependency)?
I would really appreciate any effort to help!
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Hi Omer,
You will get a lot of cell death with PMA/Ionomycin. When I most recently did proliferation assays I had to stop this at 4d and the other stimulus (PHA/TAE) at 5d for this reason. PMA activates PKCtheta and ca2+ mobilisation so is downstream of TCR/CD3, whereas PHA is a lectin which cross links all cell surface proteins including TCR/CD3 and other cytokine/costimulatory receptors.
You won't require IL2 in the medium unless you are doing TCR/CD3 +/- IL2. 
I now utilise a death marker called zombie dye to gate out the dead cells, as dead cells can lose staining with CFSE or CTV, and look like a divided population.
I hope this helps.
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We just got MUTZ-3 cell line in and wanted to check their cell surface markers -comparing undifferentiated to differentiated and stimulated etc. However we seem to be getting high levels (Aprox. 60%) of CD86 and HLA-DR on the undifferentiated cells. I would expect some expression but no where near that high and was wondering what sort of levels others normally see? Could the way we maintain the cells be in any way responsible for this? 
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 We established and characterized this cell line. CB copied our data and applied to cells of uncertain  provenance since we fused to supply them with original cells. hence,  I wonder which cells they are actually supplying. Perform STR profiling and compare them with reference profiles at DSMZ where I work.
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Does anyone have a detailed protocol of how to isolate mouse beta cells by FACS ? 
I've read in Marroqui et al. 2015 that :
"Rat β cells have a threefold higher FAD fluorescence than α cells at low glucose concentration (2.8 mM). This property, coupled to the size and granularity difference between β and α cells (β cells are larger and more granulated than α cells), allows the separation of the β and α cell fractions, with a high purity, using an average side scatter-width intensity of 170,000 units for the β cells and 120,000 units for the non-β cells."
Is this also the case for mouse beta cells ?
Thanks,
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I recommend using the MIP-GFP mouse line (https://www.jax.org/strain/006864). The original paper that described this mouse has some information on FACS sorting to isolate pure beta-cells (https://www.ncbi.nlm.nih.gov/pubmed/12388130) from this line. The complexity and size difference between beta and alpha cells holds true for mouse as well
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I am looking for a crossreactive platelet marker (preferably human) that also works on goat whole blood. So far I have been unable to find one specific for goat which is why I am trying to find a crossreactive marker from a different species. I have looked at CD11b, CD41, CD42b, CD61, CD62P so far, but without succes.
Anyone have some experience with staining platelets in goat blood?
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Hello
It is good for you to contact the technical support for Antibodies' company to make a test trial for reactivity with goat for the following antibodies:
CD41
CD11b
CD62P
I hope it helps
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I want to study the anti-inflammatory properties of a drug using RAW264.7 cell line (mouse macrophage). Most of the related publications treat the RAW cells with their drug for a short duration followed by lipopolysaccharide (LPS) stimulation to study pro-/anti-inflammatory gene expression levels.
Is the LPS stimulation necessary? If pro-inflammatory genes expression levels significantly reduce upon treatment with the drug (without LPS), can I assume the drug to be anti-inflammatory?
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Hello Divya,
It totally depends on your experimental design. If you designed and planned for any pretreatments then you have to treat the cells with your sample(s) and then co-treat with LPS. However, if you want without pre-treatments, then just treat with LPS or LPS+sample. These two ways will explain if there is any anti-inflammatory activity of your sample as well as potential of the sample in inflammation prevention. It will also provide information about inhibition of inflammatory cycles. Starting with co-treatments are more logical as it will explain the inhibition of inflammatory mediators as well as the actions of LPS.
With best
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I have some questions regarding the results of my experiments. I need inspiration and ideas to discuss these results. Hopefully someone can help me to explain this.
A short introduction of my experiment:
I isolated human PBMC and stimulated those cells with anit-CD3-AK. Additionally i applied some subances to the cells: Terbutaline, Nadolol and Dexamethasone. After 24 h incubation i measured the IL-2 concentration.
I used different concentrations of TERB, NAD and DEX.
I don´t see a concentration-dependend effect by TERB and NAD and i don´t know why. I expected that after the treatment with TERB the IL-2 concentration decreases and by treatment with NAD increases.
 Has someone an idea  what the issue could be with the above mentioned scenario?
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Hi Bianca,
A very important point is suggested in the above conversation. However, You may also try for higher concentrations of  TERB and NAD, dexamethasone to have the clear-cut effect and then try for lower concentrations. Try using a higher concentration of PBMC or you may use ConA to stimulate and go for your designed experiment. You have not mentioned the assay you are using for IL-2 measurement. you may try alternate one.
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hi
so i was told that wherever possible, it is best to use warmed reagents when doing immunocytochemistry staining steps. In the below steps, which reagents must be warmed? is it safe to say that after the fixing step onwards, it is ok to use reagents that are not warmed? i did immunofluorescence just now but only used warmed PBS in step 2. The paraformaldhyde that i use for fixing is kinda cold and i dont know if this causes the rounding of my MDA-MB-231 cells which are supposed to look kinda elongated.
1) remove media from 35mm plate that has cells growing on coverslips.
2) wash with warmed 1X PBS.
3) fix with 4% paraformaldehyde.
4) permeabilize with triton
5) Block with BSA
6) Primary antibody staining
7) secondary antibody staining
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Dear Iwan,
I would never use the solutions beyong room temperature for IHC. Usually  I use the cold PFA and rinse with  cold PBS. It works.
Good luck
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Hello,
I would like to do immunostainings on differentiated 3T3-L1 cells (adipocytes). We tried most of the "classic" protocols, fixation with either 4% PFA or cold methanol/acetone, and a lot of commercially available antibodies. If most of these antibodies work very well with other cell lines, there is absolutely no staining in differentiated 3T3-L1 cells. We suspected an insufficient permeabilization of the cells but surprisingly, one custom antibody targeting a protein from the lipid droplet membrane works perfectly...
If you thus have suggestions or protocols adapted for differentiated 3T3-L1 cells, it would be greatly appreciated.
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Hi Stephane,
If you want to 4% PFA solution for fixation,  fresh made 4% PFA solution is an important tip.
For permeibilzation (oil red O stain), 4 %PFA,  30 min at RT is enough.
For non permeibilzation (GLUT4 translocation assay by using myc-GLUT4-GFP), 4%PFA, less than 10 min, RT.  
Hope it helps.
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I isolated cells from the mesenteric lymph node of mice that were infected with Listeria monocytogenes. I had a control group, a low dose of the bacteria, and a high dose. When running the cells, a population of CD103+ CX3CR1+ cells emerged from the infected mice. I actually located them in both the colon and the MLN. The controls had mostly CX3CR1+ cells. I have looked into the literature and can't find anything about this, and thought previously that CX3CR1+ cells were CD103-.  Does anyone have any ideas? Thanks!
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Dear Haley, I think you will need to search for the literature devoted to tissue-resident memory cells, for example:
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I have tried using <40ul in a 96-well plate, which is more than enough to cover, but see differences in signal in cells in the center vs edges.
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Agree with Attila, 50uL is minimum. It's not enough to just cover the bottom because even in a wet chamber you'll have some evaporation. The first thing drying out is usually the center. Also don't take images at the edges since for some strange reasons antibodies tend to stick there... Hope that helped
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1. Why must cytospin smears be air-dried and unfixed?
2. Won't the cells morphology and contains change if the cells gets dried? Can they still be differentiated after staining?
3. Actually we don't have a cytospin in our lab and I want to spread the cells on the slides without using Cytospin. So could I fix the cells first and then get them air-dried?
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According to my experience, it's quite difficult to prepare slides from BAL fluid without cytospin. Some crust-like formation happens when the slide is left for drying on its own due to evaporation of the buffer and cells don't stick that well onto the slide. That's why cytospin is used with a filter paper to absorb extra fluid and impact only the cells to the slide. Overlapping of  cells is also a problem as you need to put a concentrated sample onto the slide. Therefore differential cell count becomes a problem with such a method.
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Historically I have always used formaldehyde fixation of cells for immunofluorescence, but lately I have had to use methanol. All of the methods I have found state to do this at -20C, but none of them say why. Just out of curiosity, and so I'm not doing it 'just because', can anyone shed some light on this please?
Thanks!
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We also typically used ice-cold MeOH fixation. I think it's a matter of intent. The MeOH denatures proteins, which may or may not be suitable for Ab staining depending upon the epitope.  Temp may provide a way to control this. MeOH also permeabilizes and thus extracts components from the cells.  Temp (with time) may regulate the extraction efficiency.
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In flow cytometry analysis of propidium iodide (PI) stained nuclei, if the cell arrest occurs in G0/G1 phase which are the most important enzymes involved in this phase?
Is there a cascade enzyme activity involved in this process? 
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Accumulation of cells in G0/G1 (cell arrest) is due to apoptotic mechanism. However to distinguish between intrinsic and extrensic pathways you need to measure caspases enzymes activity ( casp 3, 7, 8, 9) as well as detection of BCl 2 family proteins. Also as an indicator for apoptosis mechanisms, you can detect the expression of the cyclin-dependent kinase inhibitor (p21 protein).
Good luck
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Dear colleagues,
i am planning to track down the uptake and processing of a FITC-labeled antigen (e.g. FITC-OVA) by Peyer's Patches after oral gavage. Unfortunately, most of the literature deals with antigens bound to particulated matters, like nanoparticles (chitosan, etc.). Is it possible to track the uptake of a soluble antigen by Peyer's Patches as well? I plan to isolate Peyer's Patches cells and use FACS analysis or fluorescence microscopy along with T cell markers to look for Th1 or Treg shift, respectively.
Thanks in advance!
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I ve been doing the staining of peyer's patches for last 2.5 years with generalized method and getting good methods. The same method like we do with other lymphoid tissues. But there are some precautionary measures you have to take. If you need to get good results then follow the protocol of some of the recent papers where they isolated peyer's patches and you can find out them from PubMed. The method we have been doing has also provided us with good results of staining and lot more
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Dear all, I tried to induce autophagy in MEFs, but upon EBSS or HBSS starvation at different timepoint, such as 0.5, 2,4, 6, 8,18, or even in hypoxia condition, I just can't see the induction of LC-3 II, what I can see is the p62 decrease and LC-3 II is less than the 0h timepoint. there is no mycoplasma contamination and the antibody I used to detect the LC3 is from CST(4108), and I do can detect the LC-3 II induction with the choloroquire or NH4Cl. I have tried with RIPA and NP40 lysis buffer, but no positive data. could anyone have any suggestion?
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I agree that it looks like your primary antibody might be at fault here. I assume you have checked your secondary, so acquiring another primary antibody from another source such as Novus is probably a good idea.. to check transfer on PVDF, you will have to use bromoP blue instead of ponceau that works quite poorly on that type on membrane, but brilliantly on nitrocellulose. 
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Scientist has used Cleaved Caspase 3 to determine apoptosis. Also they have used Caspase3/7 assay for apoptosis. I am wondering if in "Caspase3/7 assay", the Caspase 3 and Caspase 7 are cleaved. Why Caspase 3 and 7 are assayed as combined assay as Caspase 3/7. Please clarify this confusion to me?
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Interesting question ...but answer is very simple.  The assay provides a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity and cell lysis. caspase-3 is often the primary source of cellular "DEVDase" activity, other "effector" caspases, such as caspase-7 may also contribute to cleavage of DEVD sequence present in proteins. ex PARP...it can be cleaved by both 3 and 7. so one can not say its just due to 3 or 7 its 3/7 activity. 
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i can see green fluorescence when using VSV(vesicular stomatitis virus)-GFP to infect 293T cells, but fail to see the fluorescence in primary macrophage.
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Thanks for your answer. i did not do any research about LDLR.
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does anyone know basal levels of IL6 in rats?
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Hi Dominic, some years ago I used an R&D kit with detection limit at 21 pg/ml and if I remember correctly most of my SD male, approx 3 month old controls were below detection limit (in undiluted serum). I was pretty dissapointed of wasting money then and I think I inputted the value as 0 (got wonderfull statistics...). Anyhow, VERY VERY  low.
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Do differentiated THP1 macrophages revert to normal THP1 when PMA is removed for a sustained period of time? I am looking for a transient differention followed by a return to wildtype phenotype. 
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They will indeed stay macrophage till they die after +3 days culture.
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I am looking for a recommendation for antibodies that are suitable for flow cytometry to sort based on a single human HLA receptor type (HLA-A, HLA-B, or HLA-C) but that are not cross reactive with the other species in the family.
For example, I want to sort for HLA-A positive cells with minimal cross reactivity toward HLA-B or HLA-C. Following that I would like to sort for HLA-B without reactivity toward HLA-A or HLA-C, etc.
Lastly, the antibodies should bind conserved epitopes as opposed to a specific epitope the the variable region. HLA typing antibodies that target the variable regions would not be suitable.
Can anyone recommend antibodies suitable to do this for HLA-A, B, and C?
Thank you.
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Hi Luis,
As far as I know there are no monoclonal abs with the specificities that you describe. There are however many mAbs that will recognize a small number of HLA-A or -B molecules without cross reactivity across these loci. Not much as far as anti-HLA-C and that may speak to the relatively lower immunogenicity of this locus. You can find the mAbs here:
Downside is that you will have to know the HLA type of the cells you are looking to sort.
Good luck!
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I’m planning to isolate neutrophil from buffy coat collected from healthy donors. I read about various protocols for isolation of neutrophils. I have tried ficoll and dextran methods which ends with failures. I’m really looking to find out a protocol currently used for isolation of neutrophils
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Dear Agersew, 
I have been isolating neutrophils from mouse buffy coat layer regularly using Histopaque 1077/1119 gradient. Briefly, centrifuge whole blood @ 500g, RT, 15 min. Collect the buffy coat layer and dilute 4 times with RPMI. Prepare density gradient using 3 ml of Histopaues (follow manufacturer instructions). Layer the diluted buffy coat over the gradient. Centrifuge at 700g for 30 min with no brakes mode in swing out rotors at RT. You will see a faint white band at the interface of the Histopaque gradient. Collect it carefully with a Pasteur pippete ( Dont worry if few RBCs tag along). Dilute this layer in 4 ml RPMI. Centrifuge at 350 g for 5 min. Remove supernatant and add 2ml RBC lysis buffer (Sigma). Incubate for 30 s. Add 4 ml PBS and centrifuge again at 350g for 5 min. The RBCs will be lysed and pure neutrophil population will be pelleted. Collect and observe microscopically. Hope this helps!
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I need to add IFNg to my cell cultures and neutralize it (to determine if the mechanism of my findings are IFNg-dependent).  Unfortunately there is no bovine anti-IFNg specific for neutralization. I found a recombinant bovine IFNg (143 aa, grown in yeast), and I found a bovine anti-IFNg against a 143 aa recombinant IFNg grown in yeast, but it is specific for ELISA and WB. Would that work to neutralize?
Someone from technical support recommended me to buy a human anti-IFNg (much much more expensive), but it doesn't really make sense to me... SOS!
If the anti-IFNg I found works to neutralize, can I confirm the neutralization with ELISA (using the rBoIFNg + anti-IFNg after incubation)?
Thanks!!
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You could as well try human ifng that should work on bovine cells. Then neutralize with anti human.
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I am having difficulty detecting LC3 from Jurkat cell lysate. I am using a 14% SDS-PAGE gel recipe which works well detecting LC3 from CACO2 cell lysate. The lysate samples are kept in the freezer, brought out and kept on ice when required. The lysis buffer used is Triton-X100 with added protease and phosphatase inhibitor. The appropriate amount of lysate is added to the sample buffer and boiled in a heat block for two minutes. I load 20µg/µl in each well and the gels run fine (120V). The gels are then transferred on an iBlot (Invitrogen) which works well. The membranes are then blocked in 5% BSA for two hours followed by an overnight incubation at 4ºC with primary LC3 antibody 1/1000 dilution (Cell Signalling). The following day the membrane is washed in TBST (1x) for 3 x 10 minutes followed by incubation at room temperature with the secondary antibody for one hour. The washing step in TBST is repeated before the membrane is exposed to ECL substrate (Bio-Rad clarity western ECL) and the membrane is placed on a Chemidoc (Bio-Rad) for band exposure.
Sometimes I get LC3 bands but they are quite weak. I am specifically looking for the two LC3 bands (flux between LC3 I and LC II), which are sometimes present but again are very weak. If anyone can suggest anything, I would be grateful. I have attached an image of an example developed membrane to give an idea of the weak bands.
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Christopher I am attempting to detect LC3 in trophoblast cells and note that you use the iBlot transfer system as well - may I ask which program or settings you used for the transfer?
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Our lab recently did a test run mixed lymphocyte reaction (MLR) using normal human PBMCs.
Medium composition was HyClone RPMI-1640 +10%hiFBS +P/S +Fungizone +2.05 mM L-glutamine.
The feeder cells were treated with mitomycin C at 50ug/ml for 1 hour to render them non-proliferative.
DPBS was put in the space between wells to help minimize effect of evaporation and edge effects. 
Cells were grown for 6 days at 5% CO2/100%RH/37'C. Incubator logs show no deviations.
Excess stock from which cells were taken was plated in a six-well plate in the same media and grown alongside the MLR plate. These cells are still alive. The 96-well plates used for MLR are brand-new. There was no sign of any bacterial, fungal or yeast contamination. 
MLR is new to me, so it may be that I'm missing something obvious. What's going on here? Why are all my cells dead in the MLR but not in my 6-well plate?
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- If you are not obtaining the cells form an infected site of a biopsy then remove fungizone.
- Be sure that you extensively wash MitoC away from the target cells before adding them into the MLRs.
- Prefer U-bottom 96-well plates and adjust the cell number accordingly.
- 4-day-long incubation can also work.
These are basic recommendations... you may also like to think about adding supplements like IL-2 or non-essential amino acids etc. to support cells' viability.
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Hi all, when I isolate the immune cells from the lamina propria, after the first media digestion (RPMI 1640, 5mM EDTA, 20mM HEPES, for 20 minutes, 37 C ), I found that some intestine becomes white, and we can't get immune cells from those intestine, Does anybody know the reason, and any strategy to deal with this situation? Thank you so much.
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Definitely try the  5 or 10% FBS first because any incubation step of digesting cells you extract from mice you killed should have FBS as a digestion solution component. Otherwise without serum, you can easily lose like a large portion of viable cells (anywhere from like 20% to over 50%) in your sample. Well they'll still be there but they'll be dead.
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What kind of reagents do you use to mature dendritic cells (after isolating monocyte from blood bank buffy coats and differentiating them into immature DCs using GM-CSF and IL-4)?
There are a lot of different methods used in the literature by good labs, including
1.     TNF, IL1B, PGE2, IL-6 (“gold standard”)
2.     IFN, Monophosphoryl lipid A (MLA)
3.     TNF, IL1B, PGE2, IFNy
4.     TNF, IL1B, PGE2, MLA
5.     LPS, IFNy
6.     TNF only
7.     LPS only
8.     TNF, IFNy
9.     TNF, IL1B, IFNy, IFNa, PolyI:C
And some researchers use R848 etc, etc
We are mainly aiming for maximal IL-12 secretion by the DCs and also allostimulatory capacity. Obviously maximal migratory capacity would be great as well, but at the moment this is a less important consideration for us. Therefore we are thinking of something like TNF, IL1B, PGE2, IL-6, MLA, or TNF, IL1B, PGE2, IFN, MLA, or something like that.
We are trying to create immunogenic DCs rather than tolerogenic DCs.
Thank you
Dominic
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I dont know what is the best way but i know it is important to choose the best Medium for your aim. In my experience the output of some MoDC´s cytokine are better in serum free Medium like X-vivo 15 than in RPMI1640.
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Hello
We want to estimate gut inflammation and oxidative stress. For certain reasons, we can't use gut tissue in human disease model. Can we use stool samples to estimate inflammation cytokines and oxidative stress? Alternatively, I read somewhere that there are few cytokines, which are only associated with gut inflammation and can act as indicator of gut inflammation only. They can be studied in plasma. Are there existing similar oxidative stress indicator for gut as well?
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HI Dear 
it is suitably to use liver biopsy to used them in IHC. the best marker is 8-OHDG. Also you can check them using ELISA kits but the collection and preservation time of serum must be not more than 6 hours.
BR
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Thanks
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Hey
Check  Links below  PLZ
National Center for Biotechnology Information
by AH Badawi - ‎2013 - ‎Cited by 6 - ‎Related articles
Aug 1, 2013 - In EAE and MS, the activation of inflammatory CD4+ T cells is ... In this case, the efficacy of MOG-BPI was evaluated in PLP-induced EAE, and the .... a protocol similar to that reported previously (Youssef et al., 2002). ... Splenocyte Proliferation Assay ... One group consisted of mice that had no EAE induced.
National Center for Biotechnology Information
by E Bettelli - ‎2003 - ‎Cited by 492 - ‎Related articles
MOG-specific transgenic T cells are not deleted nor tolerized and are functionally .... For proliferation assays, splenocytes (5 × 105 cells/well) were cultured for 72 h in ... Three different protocols were used to induce disease in transgenic and ... 2, A and B , spleen cells from naive 2D2 TCR transgenic mice with no sign of ...
Hooke - Protocols - Cytokine Production Induced By T Cell Recall ...
hookelabs.com › Protocols
Hooke - Protocols - Cytokine Production Induced By T Cell Recall Response In Vitro. ... Full product catalog · EAE induction Hooke Kits™ · CIA induction Hooke Kits™ ... MOG35-55 in TC Media, 100x (cat. no. DS-0111) ... FBS for T cell culture, 100 mL (cat. no. .... IL-2 is consumed by the proliferating T cells in these cultures.
Best regards
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I know experimentally the percentage of CD4, CD8 and NKT cell equivalents I 'm getting from my guinea pigs but would like to know if it is comparable to murine and human, or if substantial proportional differences exist.
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Hi Julianna,
Here is a reference on relative  % within the mouse spleen from Stemcell.
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I'm differentiating THP-1 in macrophages by PMA treatment. Protocol is PMA 100ng/ml treatment fr 24 & then rested in PMA free media for next 24h?
After 48 hours what percent of THP-1 cells are differentiated into macrophages or how can I calculate it ?
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Hi Hafij,
I used to seed approx 0.5 X 10 cells/per well in a 6 well plate. I always get a good differentiation with this numbers. 
For 24 well plate seed approx 0.1 X 106/well in 1 ml of medium.
Its advisable to differentiate them for 24 hrs in the presence of PMA and rest them for the next 24 hrs without PMA.
With these number of cell, i always get good infection and similar results every time.
Best of luck....
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There are many markers to look at to determine the effectiveness of a t-cell in immune reconstitution and gvt/gvhd responses prior to mouse transplantation such as cytokine, proliferation, expression of some cell surface markers.
But I think it would be pretty cool if we could see and compare the effectiveness of t-cells cd4/cd8 in fighting tumor in vitro. Wouldn't that be a nice way to see how a t-cell would respond in transplantation for a mouse with tumor growth?
But is it as simple as mixing some apc's and tumor cells with cd4 and cd8 cells? Are there other conditions I need to make t cells kill tumor cells in vitro?
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It depends with whether your T cells have been primed against the tumor antigen before. We find we can get a recall response in that situation without additional stimulation needed. Just add tumor and T cells in one to one ratio. But if they are not primed you may require additional cytokine stimulation.
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Using a CD-25PE CD4-PE stain, same sample, two different cytometers, gives different results, a 50% dp population or an 80% population.  Both cytometers were calibrated with CST beads and compensation was done correctly as far as I can tell. I attached the plots
Any ideas?
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I have not used a Canto, but have lots of experience with the Caliber and an LSRII.  I would imagine that a newer machine would be made to be at least as good as an older machine, Then again, selections of filter etc that allow for more channels can affect the overall sensitivity and how parameters are displayed.  I've seen sensitivity and displays change with different filter set-ups.  
Signal to noise is simple for you to calculate:  Gate on the CD4+ and determine the Mean Fluorescence Intensity for CD25 expression.  Do the same for the isotype control (don't gate on CD4 if you don't have the CD4 vs isotype control).  Then simply divide the signal (CD25)MFI by the noise (isotype)MFI to get a ratio.  A higher ratio indicates greater sensitivity.  
Your data display differences, I think, is your major issue.  I'm hoping the S:N calculation will let you see which machine is more sensitive.  I have to say that I think the display of the Caliber data appears to be better for resolving the CD25+ subpopulation, but maybe this can be fixed on the other machine by changing the bi-exponential/log collection parameters.
My point about gating affecting the frequencies is not about the gates used before the plots you showed but, rather, the actual quadrants used.  My point is that CD25 expression is a continuum.  Moving those quadrant gates just a little closer or further away from the controls can have a big affect.  If your using FlowJo, you could use some of their algorithms to calculate the percent positive by subtracting controls from your CD25 staining on histogram plots.  That way no lines are drawn by hand and you'll have the percent positive calculated by math.  Having said that, I rarely use this technique in the real world because I find it to be more sensitive than I think is real.  However, it is unbiased – or consistently biased.
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I am interested to measure inflammasome activity in tissue. I wonder if Caspase-1 activity assay which is often used in vitro is suitable for tissue samples?
Many thanks.
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Dear Chung-Wei,
The Immunchemistry Technologies FAM-FLICA fluorescent caspase-1 probe can be used to detect active caspase-1 also in frozen tissue sections; see the link below and the attached manual for a tissue section staining protocol suggested by the manufacturer (section 23).
The probe is based on a caspase-1 peptide inhibitor that recognizes and binds to the active site of the enzyme, forming an irreversible covalent bond and blocking further enzyme activity. Thus, this reagent should detect only the active form of caspase-1 in tissues, which is a direct indicator of inflammasome activity downstream the activation of the various different inflammasome receptors (NLRP3, NLRP1, NLRC4, AIM2, NLRP6, PYRIN and so on.).
You can also use an antibody against active caspase-1 p10 or p20 subunit, but this is more reliable in western blotting of tissue extracts rather than in IHC, as you can confirm that the antibody truly recognizes only the active p10/p20 subunit without cross-reacting with the inactive proform.
Best regards,
Kristiina
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Hello everyone! Hope everyone's okay!
A thought has been crossing my mind from a quite a few days.
The gene that imparts heat resistance capabilities to the organism Thermus aquaticus (Taq), can it be incorporated into other organisms' genome through available techniques?
Or is it possible, that the same sequence is present in other organisms such as humans or plants, but is inactive and be activated through some stimulus or a pathway?
Basically, the main point is can the heat resistance gene of Taq be made to express the same abilities to other species e.g. humans and plants?
Need your thoughts.
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There is no single "heat resistance gene". All genes in thermophilic organisms have evolved to have higher thermal stability. This increased stability can be observed in highly purified proteins. Individual thermophilic genes can be expressed in mesophilic organisms, however, they might not work as well at the lower temperature these organisms grow at.
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I'm trying to analyze liposomes using flow cytometry with a FACSCalibur, with little experience with either. I often suddenly lose signal.
Signal eventually resumes after copious flushing with bleach and water, and rebooting the cytometer and CellQuest. This loss of signal doesn't happen to colleagues who are looking at more conventional cell preparations. Is it something I'm doing, or the liposomes?
Liposomes are about 400 nm, pegylated.
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I'll let you know. Thanks for your interest.
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Using LPS stimulation, I could easily detect iNOS induction from Macrophage (Ma) or Microglia (Mi) after 6 hr, 16hr by qPCR.
Even though I detected the translocation of NF-kB p65 into the nucleus after 30 min when challenged cells with TNFa (20, 100ng/ml) or IL-1b (20ng/ml).  I could not detect the iNOS induction after stimulation with these cytokines after 6 hr, 16hr by qPCR.
Does anyone knows the reason and how I should change to get induction of iNOS ?
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In my case, I stimulated BV2 microglia cell line with LPS (100ng/ml) and detected iNOS (by Western Blot) and NO ( by greiss assay) after 20 hour. at this time point, I get clear WB signal as well as significant increase of NO production. 
I also work on primary microglia, astrocyte and Raw264.7 macrophage as well. I think they are just different on the sensitive but not much.
you can try to incubate cell longer ( if the concentration of your stimulator is within middle range). I saw many of other paper they often try to measure iNOS and NO after 24 hours.
Good luck
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