Questions related to Cellular Immunology
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.
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?
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.
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
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
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.
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.
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?
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.
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?
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.
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.
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
- Antihistamines administered early in infection may reduce excessive cytokine proinflammatory storms.
- Zinc supplementation of population
- Introducing anti-inflammatory cytokines and/or monotherapy blocking IL-1 cytokine or receptor, inhibiting IL-1 may inhibit the inflammation.
- IL-4,6,10,11 and 13 are anti-inflammatory cytokines
- IL-1 receptor antagonists
- Chloroquine, the antimalarial drug that inhibits lymphocyte proliferation. As well as anti-viral activity. US have approved this therapy.
- 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
I have used the following markers in a flow cytometry panel to assess mouse samples (7 days old mice and 30 days old):
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!
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?
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
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.
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.
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?
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.
Is it possible to used the rabbit polyclonal antibodies as an antidote for human and is that legal to be injected to human?
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 ?
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!
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!
Here is the paper:http://www.ncbi.nlm.nih.gov/pubmed/20497038
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.
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?
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?
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?
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.
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?
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!
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?
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 ?
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?
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?
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?
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
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.
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!
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.
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?
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?
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?
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!
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?
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?
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.
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.
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?
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
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)?
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.
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?
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.
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.
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?
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.
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 ?
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?
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
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.
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.
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 ?