Cellular Immunology - Science topic

Isolating lymphocytes from a mouse spleen involves several steps:

1. Spleen collection: Harvest the spleen from a mouse and place it in a sterile container with cold PBS (phosphate-buffered saline).

2. Spleen dissociation: Mechanically dissociate the spleen into a single-cell suspension using a tissue grinder or a syringe.

3. Red blood cell lysis: Remove red blood cells by adding a lysis buffer (e.g., ACK lysis buffer) and incubating for 5-10 minutes.

4. Centrifugation: Centrifuge the cell suspension at 300-400 x g for 5-10 minutes to pellet the cells.

5. Cell washing: Wash the cells with cold PBS to remove any remaining debris.

6. Density gradient centrifugation: Layer the cell suspension over a density gradient medium (e.g., Ficoll-Paque) and centrifuge at 400-600 x g for 20-30 minutes.

1) I think the cells should recover within 1-1.5 week. After 2-3 weeks they still can be rescued, but they need to be carefully monitored, how they are growing on the next weeks and how they behave in the functional tests. In your case something went wrong, since the viability is around 40%. Have a closer look on your freezing/thawing procedure, check the incubator.

2) I would be careful with trying to supplement more frequently with IL-2, in my opinion it could mess up with the activation process. You can of course experiment with such supplementation but I would keep the tests in the extra well, if you have enough cells to have an extra one. Instead, I would increase the percentage of serum during 1-2 passages (5-10% more than you usually use) and observe the effects. According to my experience, there is a good chance this may work, we were boosting some lympocyte lines with extra serum to increase the viability.

3) Check if there is no mycoplasm contamination. You can also pippet the cells every day to allow reforming the aggregates (and therefore getting rid of the dead cells from the inside of the aggregate). Also, play with splitting ratios. It can be especially important during the first passages after thawing, I would recommend to split the cells in a higher density than after 1-2 weeks of culturing them, they need to "wake up" and reach their max doubling rate, and sometimes it takes some time.

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.

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:

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

Fcs alternatives for HL 60 neutrophil like cell line is FPS (slightly different).

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,

Dear Abdelwahab Zemam

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.

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.

I think yes.

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.

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

Here is an excellent video explanation on the concept of signal width, and how it is affected by clumping of cells.

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.

Before reaching to any conclusions one need to know values of CD3, CD 56/16.

On what population did you gate CD 45RA and CD 45RO ( on CD 3 or cd4)?

What are it's Immunoglobulin profile and total /differential counts?

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.

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.

Dear Felix Kretzschmar

concerning your question:

In brief, for the gating strategy, please see attached:

https://www.pnas.org/content/pnas/suppl/2015/08/14/1506254112.DCSupplemental/pnas.1506254112.sapp.pdf

Macrophages are often defined as CD11b⁺/F4/80^high, whereas monocytes are defined as CD11b⁺/F4/80^dim, which is a bit arbitrary, without additional markers.

The supplementary figure is from the following paper in PNAS:

https://www.pnas.org/content/112/35/11001.long

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

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.

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.

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

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!

Dear It is possible to stimulate the production of antibodies Post infection.

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.

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

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

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).

following

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).

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.

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.

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.

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.

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.

Hey Robert, did you ever find an answer to this question?

So many thanks for your valuable recommendations. They did help alot.

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.

 Thanks Dr. Ponnuraj.

Enzyme-linked immunosorbent assay (ELISA) based methods are the most efficient in quantifying the level of antibody in a given sample.

Dear Bruce

Thank you for your kindly reply.

Best regard

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. 

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.

 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.

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:

http://www.abcam.com/cd41-antibody-co35e4-ab23615.html (already anti-goat)

http://www.abcam.com/cd41-antibody-ab63983.html (predicted)

http://www.abcam.com/integrin-alpha-2b-beta-3-antibody-co35e4-ab81434.html (already anti-goat)

http://www.abcam.com/cd11b-antibody-ab75476.html (predicted)

http://www.abcam.com/cd11b-antibody-ab62817.html (already anti-goat)

http://www.abcam.com/cd62p-antibody-pselko27-ab54427.html (already anti-goat)

I hope it helps

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

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.

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

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.

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

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.

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.

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

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

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. 

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. 

Thanks for your answer. i did not do any research about LDLR.

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.

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!

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!

You could as well try human ifng that should work on bovine cells. Then neutralize with anti human.

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?

- 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.

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.

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.

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

Hey

Check  Links below  PLZ

www.ncbi.nlm.nih.gov/.../PMC41391...

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.

www.ncbi.nlm.nih.gov/.../PMC21939...

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

Hi Julianna,

Here is a reference on relative  % within the mouse spleen from Stemcell.

Hi Hafij,

I used to seed approx 0.5 X 10⁶  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....

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.

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.

I'll let you know. Thanks for your interest.

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

Aicha, I also encourage you to submit reviews of the 4 antibodies you used to pAbmAbs Antibody Reviews (http://pabmabs.com/wordpress/?page_id=5). This is a good way to help future researchers!

It's a clever and probably important question.  I won't pretend to know an answer, but I'll say whatever it is that you're trying to do with this information, I'm excited about it.  Good luck & I hope others will have something smarter to say. 

Hi Yanick,

  are ur stem cells proliferating in your control settings? If they are it could explain the loss of density of class I molecules at the surface. and I guess your IFNg treatment is inhibiting this proliferation.

The suggestion of Constatin can also be the cause of your observation.

Another way of doing the experiment is by measuring the de novo expression of class I molecules by doing a pulse chase with marked methionin. This will allow you to measure the actual new levels of MHC class I expression post stimulation with IFNg.

You could also measure mRNA levels by qRT PCR, it would give an indication about the stimulation but for real protein levels u should measure proteins.

I hope this helps

Good luck

G.

The HETRASE trial randomly assigned 319 patients with sepsis to receive low dose intravenous heparin (500 units per hour) or placebo for seven days. The