Hi Felix,

PHA and PMA/Iono act on cells in a quite different manner.

PHA (Phytohaemaglutinin) is a lectin, binds to the sugars on glycosylated surface proteins, including the TCR, and thereby crosslinks them. So with PHA you get TCR crosslinking and signal 1 (and possibly also signal 2 via crosslinking of co-stimulatory molecules) required for T cell activation. These processes happen on the cell surface and might therefore involve a variety of signalling pathways.

PMA on the other hand is a small organic compound which diffuses through the cell membrane into the cytoplasm, where it directly activates Protein Kinase C (PKC) omitting the ‘need’ of surface receptor stimulation. Ionomycin, a calcium ionophor, is used in addition to trigger calcium release which you need for NFAT signalling.

If you just need to do a brief ex vivo stimulation, 5h PMA/Ionomycin/GolgiStop is pretty standard and should be okay. For a more physiological (or longer) stimulation, antiCD3 plus either antiCD28 or APC would probably be better. I personally don’t really use PHA, I think it’s too messy. Your signals could be coming from any glycosylated surface molecule.

Maybe this helps a little.

Hi Felix,

PHA and PMA/Iono act on cells in a quite different manner.

PHA (Phytohaemaglutinin) is a lectin, binds to the sugars on glycosylated surface proteins, including the TCR, and thereby crosslinks them. So with PHA you get TCR crosslinking and signal 1 (and possibly also signal 2 via crosslinking of co-stimulatory molecules) required for T cell activation. These processes happen on the cell surface and might therefore involve a variety of signalling pathways.

PMA on the other hand is a small organic compound which diffuses through the cell membrane into the cytoplasm, where it directly activates Protein Kinase C (PKC) omitting the ‘need’ of surface receptor stimulation. Ionomycin, a calcium ionophor, is used in addition to trigger calcium release which you need for NFAT signalling.

If you just need to do a brief ex vivo stimulation, 5h PMA/Ionomycin/GolgiStop is pretty standard and should be okay. For a more physiological (or longer) stimulation, antiCD3 plus either antiCD28 or APC would probably be better. I personally don’t really use PHA, I think it’s too messy. Your signals could be coming from any glycosylated surface molecule.

Maybe this helps a little.

I am absolutely agree with Susanne.

I also agree with Susanne.

Moreover, if you need to get an antigen specific response you can stimulate with antigen plus anti-CD28 for approximately 48-72 hours and add brefeldinA (and monensin) for the last hours.

If the response is too low you can boost the response restimulating with Ag+antiCD28 and IL-2 for 3 more days and after one week you block Golgi and perform the intracellular flow analysis.

Dear Felix,

I have an experience with PMA(10ng/ml) plus Ionomycin (1um) and also with PHA(0.3 ug/ml) on human PBMC activation for 48hr.I have measured different types of cytokine including IFN-gamma in cell couture supernatant by Elisa .in this experience we also measured the proliferation rate of lymphocyte by LTT.

PMA+ inomycin activated T-cell more strongly than PHA from viewpoint of all cytokine production and lymphocyte proliferation

Good luck

Amrollah

As Sussane mentions, PHA is messy - especially when you use it for stimulating cells to be acquired via flow cytometry. PMA/Ionomycin is less messy but it causes masking/down-regulation of the CD3/TCR - which can make it difficult to identify your Tcell population via flow. If you can, try SEB/SEA (staph enterotoxins), they are superantigens that (in a nut shell) crosslink the MHC-II and TCR (stimulating >75% of all Tcells) causing Tcells to proliferate and produce many different cytokines (including IFNg and TNFa). I've found that SEB/A works best as a positive control antigen for flow - and should serve well to monitor Patient Tcell exhaustion (I used to do this for HIV-patients many years ago using PHA and recall antigens - before flow and intracellular cytokine staining). The problem with SEB/A is that some institutions don't allow you to use it (or make it very difficult) because it is considered a Select-Agent - although, if you keep

Hi all. I have related question:s how do you deal with phenotypic changes occurring after pma/iono stimulation? Have you experienced a shift in background fluorescenve in lymphocytes of treated vs untreated samples?

Background shifts due to stimulation are largely due to increased autofluorescence, caused by the stimulated cells becoming larger (a result of cellular activation). Using an appropriate control is how you "deal" with these changes - in this case, you would make sure you had a matched isotype control to help determine if the increase was due to increased autofluorescence or an actual increase in signal, something that your biological control (unstimulated vs. stimulated) can't account for...anytime you are doing flow, it is very important to include multiple controls (FMO, biological, isotype controls) - they each serve important function.

Thank you all, especially Susanne and Charles, for your helpful answers. They were more informative and concise than everything I found in the literature so far.

Hi Felix,

I also agree with Susanne and Charles, however I will add a couple of points more: PMA/Ionomycin will get you a very strong and TcR independent activation of everything including T cells. PHA on the other hand will get you activation through many surface receptors including TcR and this can be very messy. The closest T cell mitogen to Ag specific TcR activation are the anti-CD3/CD28 beads or Superantigens (SEA-SEB-TSST-SEE...). The beads will allow you to activate over 80% of your T cells without any need for antigen presenting cells (APCs). The superantigens (SAg) however need to be presented by MHC II bearing APCs. This also contributes to their closeness to an Ag specific activation of T cells.

If I have to do your experiment I'd use the beads for a very restricted TcR induced activation of the T cells. On the other hand if you're trying to recreate a physiological model of T cell activation the cross talk between the APCs and T cells might be important than I'd look into the SAg presentation.

Good luck

George brings up a good point - given you're looking at functionality versus exhaustion, you may want to use both a superantigen (APC-dependent) and anti-CD3/28 beads (TcR-independent stimuli) to cover APC dysfunction-related and Tcell-dysfunction-related exhaustion. Though I would advise making your own anti-CD3/28 beads, because they are expensive (at least here in the states).

Hi all, really valuable information! What do you think about using ConA for non-specific T cell stimulation?

Hi all. This turned out to a very interesting exchange. Con A also needs antigen processing and presentation. As Charles mentioned above, to use both systems: APC-dependent versus independent, you can assess whether or not antigen-presentation is involved in T cell dysfunction, Charles, you may want to check Dako, which was an Scandinavian provider of reagents. Not sure it was bought by another company, probably they developed the system of bead-bound antibodies back in the 90's.

Hi,

Consider also that PMA/CI suppose to activate the cells that are already primed to produce the cytokine.

Dear Felix

I have experience with LPS as stimulator. My problem is the quantification.This method distinguishes between different cell types. However the quantitative distribution is not determined exatly. The same problem is in the case of the measurement of the intracellular cytokine production. I am missing the exactitude. What is your opinion, what is your experience?

with best wishes

eva

Dear Eva,

my experience with LPS is limited to its use for the maturation of dendritic cells, I have never used it as a stimulator of T cells. Maybe somebody else in this forum can better help you with this questions.

With best wishes

Felix

Dear Felix

Thanks for your answer.

Are you satisfied with the quantitative evaluation of the method: Measurement of intracellular cytokines? Not me... This is not exact.

best

Eva

Hi, Eva and Felix. As usual, the correct method to use is dependent on the biological question you have. Intracellular cytokine staining (ICS) can give you a number of informations such as: what's the cell type that respond to a specific stimulus, by the production of a specific cytokine, in mixed populations (such as PBMCs, or splenocytes)?. If you have quantitatively measured a response i.e. from PBMC, and need to identify responding cells, then the correct use of flow cytometry, optimized to detect cytokine, can help you. In other occasions, flow cytometry can be one of the most refined techniques to measure responses. One example: antigen specific lymphocytes are very rare in the circulation, so that the measure of bulk cytokines from supernatants is not feasible. The definition at the single cell level of flow cytometry once again helps you in the quantification of the response, measured by the combination of the frequency of cytokine positive cells and of the median fluorescence intensity of positive cells. Also in this case, the correct evaluation of both the percentage of positive cells and of their MFI has to be done by correct subtractions and controls. Some references:

1) Shooshtari P, Fortuno ES 3rd, Blimkie D, Yu M, Gupta A, Kollmann TR, Brinkman

RR. Correlation analysis of intracellular and secreted cytokines via the

generalized integrated mean fluorescence intensity. Cytometry A. 2010

Sep;77(9):873-80. doi: 10.1002/cyto.a.20943.

2) Kutscher S, Dembek CJ, Allgayer S, Heltai S, Stadlbauer B, Biswas P, Nozza S,

Tambussi G, Bogner JR, Stellbrink HJ, Goebel FD, Lusso P, Tinelli M, Poli G,

Erfle V, Pohla H, Malnati M, Cosma A. The intracellular detection of MIP-1beta

enhances the capacity to detect IFN-gamma mediated HIV-1-specific CD8 T-cell

responses in a flow cytometric setting providing a sensitive alternative to the

ELISPOT. AIDS Res Ther. 2008 Oct 6;5:22.

3) Tanaka Y, Ohdan H, Onoe T, Asahara T. Multiparameter flow cytometric approach

for simultaneous evaluation of proliferation and cytokine-secreting activity in T

cells responding to allo-stimulation. Immunol Invest. 2004 Aug;33(3):309-24.

Many thanks Mario. I will read the publications. In my experience the evaluation by percentage is not exact regarding the intracellular cytokine production.

best

eva

Hi Felix & Eva, to the best of my knowledge, LPS is a TLR-4 and CD14 ligand (among others, I'm sure). The main receptor for bacterial LPS seems to be CD14, so if you are using that to stimulate T cells, make sure you are just using T cells. It may also be important to note that soluble CD14 is present in the circulation and has been shown to inhibit T cell activation. TLR-4 activation has been shown to stimulate autophagy by macrophages, and to affect a pro-inflammatory response in T cells (makes sense considering the intracellular domain for TLR-4 is a homologue of the IL1r intracellular domain). The TLRs are present on many cell types but seem to correlate to innate immune pathways. As such, using a TLR ligand alone to stimulate T cells will probably give you a more innate, pro-inflammatory cytokine response profile than using a more broad T cell activating agent, like anti-CD3/28, PMA/Iono or PHA. Autophagy stimulated by TLR-4 activation may also lead to erratic, inconsistent cytokine staining.

Felix, regarding your T cell activation protocol, I agree with Susanne. The more physiologic of a response you want, the closer to physiologic conditions you will have to get, and it will likely give you the best results, although at a higher monetary and temporal price than other protocols. I have used pre-loaded APCs, anti-CD3/28, PMA/Iono and PHA in the lab to stimulate T cells. A roadblock to you using a prime-stimulate method is that you do not have a specific antigen to load and using a random antigen will probably get you many naive T cell responses (and not enough memory/effector responses, unless you are priming with superantigens, like Georges mentioned). I would suggest using a magnetic cell sorter to separate your naive and other T cells, and testing them separately using a variety of methods, to see what is most effective per cell phenotype, given the pleiotropic responses of different cells to the same stimuli. Anti-CD3/28 is an easy way to stimulate T cells in my experience, and it works great too (also expensive, though). PMA/inono is also good, but keep in mind these molecules are toxic to cells, and I believe they bind their targets irreversibly, so watch out for AICD and keep your incubation period to the minimum necessary. PMA/Iono also seemed to work best for ELISA for me; anti-CD3/28 best for flow (just make sure your stains don't bind the same epitope of CD3). As Susanne mentioned, PHA will probably give you a rather messy result, for the reasons she mentioned.

In sum, given your situation, I suggest PMA/Iono (of the two methods you mentioned; antiCD3/28 is best for this specific case in my opinion). The methods of action of these drugs are well-studied, they work well and the response you get is more indicative of general activation. Just be sure to mix the PMA/Iono well in the cultures...I suggest making a stock dilution so you can add maybe 10ul to each well in a 96-well plate, so you have enough volume to mix evenly and are not adding 1-2ul to each well. I hope this helps!

Cheers,

MJ

I have not read above answers and don't know if people mentioned this. If you are using PMA/Iono protocol for human T cell stimulation just be aware that CD4 is downregulated on them. You might not be able to gate on CD4+ T cells unless you perform intracellular staining for CD4 as well.

Dear Jaba, I agree with you about the downregulation of CD4 after PMA+Iono activation.

In order to bypass this drawback you can gate cells in lymphocyte gate (FSC/SSC) + CD3+cells and look for CD8- (almost all CD4+ T cells) and CD8+ (CD8+ T cells) + intracellular cytokines. We have done this looking for cytokines and a cell cycle marker such as Ki67, with good results.

I want to develop a methodology for intracellular staining of cytokines (IL-2/INF-gamma/IL-4/IL-10) in Jurkat E6.1. Does anybody have an advice for this aim.

Thank you

I have not knowledge or I have not found, antecedents of intracellular staining of cytokines in Jurkat T cells and which one inducer most suitable.

Thanking you in advance for your responses

Dear Felix,

In my experiment, PMA/Iono stimulated much more stronger than PHA the PBMC prepared from healthy individuals as well as patients with T1D .we determined the IFN-y secreting cells activated by PMA/Iono or PHA by ELISPOT and analyzed the supernatant by ELISA to assess this cytokine.

Good luck

Amrollah Mostafazadeh

About the CD4 down regulation, one option is also to stain for intracellular CD4 as it is internalised if I'm not mistaking. Enjoy! :)

Thanks to all, its very useful information. I am working with pure activated CD4 T cells (CD3/CD28 MicroBeads, Miltenyi), I want to evaluate cytokine production by Elisa. I wish to know if I can collect direct supernatant from cultures or re-stimulate with PMA/Iono?  

I agree with Susane

 The mode of action of lectins is very different from small molecules like PMA and ionomycin. We regularly use this combination in the presence of Brefeldin for a 4 hr stimulation to study intracellular cytokine levels .  For proliferation assays we use anti CD3 /CD28 or specific antigens.

Just wanted to add this paper as a nice reference for comparing PMA/Iono with PHA:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC96054/

Conclusion - it depends on what cytokines you are looking for. 

Hello all,

This is very useful information. Wondering if I stimulate my T cells with anti CD3/Cd28 then to detect cytokines would you add Golgistop/plug/breferdin and additional antiCd28/49d co stim or would you not add a few of these. 

Thanks

Krupa, the CD3/CD28 stimulation is pretty potent. Adding additional stimulation may overload the cells. If long enough, they would die off. Safer to be careful with this approach.

Felix, you got plenty of info in the preceding replies. One important detail between your decision on PHA or PMA/Iodo, these two methods get processed using different pathways. PHA requires antigen processing (and antigen-processing cells) before activating T cells, whereas PMA/Iodo directly acts on T cells. If you have purified T cells or cell lines, PHA wouldn't work. So, it depends on what your system is and what you need to address.

Thank you all for this great discussion. I just have little question. Can we find an increase in the expression of the transcription factors (like T-bet , GATA3 or FOXP3) in T cells treating for 5 hours with PMA+ionomycin?

I have treated T cells with PHA for 48 hours but i did not find a good increase in the expression of these transcription factors .. do you think i can have an increase using PMA+ionomycin?

Thank you all

Ousama, I never tried myself detecting expression of those factors, but I'd expect to see an increase since cytokine secretion is certainly increased.

Hi Li, 

regarding question 2, I was asking myself the same question, so I performed an experiment with three different conditions to look at IFNG expression in Patient samples:

1-with no manipulation (naïve cells) 

2- only GolgiStop added

3- PMA+Ionomycine+ GolgiStop

I found some IFNG expression in naïve cells, which changed very slightly by activation or proton pump inhibitor.

Since the aim of my study to compare IFNG expression between healthy and patients samples, and knowing I have seen no INFG in healthy controls and increased in patients, that was enough evidence for me to use Naïve cells directly.

However, I found no reference for my method as all papers I have seen activated and use proton pump inhbitors, so I might refer to the optimization experiment I did. please let me know if u find anything in literature

Hope that helps

Fatma

Hi Dr. Villacres,

You wrote that "PHA requires antigen processing (and antigen-processing cells) before activating T cells". The presence of APCs is clearly required in normal conditions, but could you please explain a bit what you mean by "antigen processing"?

I was under the impression that PHA is capable of stimulating T cells without intracellular processing and subsequent presenting on MHC class II molecule on the APC surface (as it can also stimulate MHCI-dependent CD8+ T cells and the stimulation is independent on MHC restriction), and that APCs are required to provide the required co-stimulatory signals, not to process the antigen per se. Am I getting this wrong somehow?

Hi everyone,

I was searching for papers related to PHA activation of Jurkat cells and the flow cytometric analysis of CD69 marker on the activated cell. But unable to find a good literature. Kindly suggest me a good paper related to it.

Thanks.

Hello,

If I want to follow marker internalisation, should I activate jurkat cells with PHA, PHA+PMA or PMA+iono?

Would PHA (1ug/ml) induce cell death of jurkat cells?

Thanks,

Vince

Hi everyone,

we stimulated whole blood culture from healthy person with PMA/ionomycin and also inactivated influenza A virus. IFN-g and IL-2 levels decreased as against PMA/ionomycin stimulation in absence of inactivated influenza. What is the reason for that? 

Thanks for your help

Influenza virus maybe has immunosuppression effect ? 

My personal experience is antiCD3/28. This works amazing for T cell stimulation. We in lab have been using this method periodically to stimulate total PBMCs and this enriches T  cell stimulation. However as mentioned above this is an expensive way to go with. PMA/Iono/Golgi stop is toxic but again highly effective way to test production of IFNg. I would also go ahead and look for cell surface antigen, CD107a. They work out well 

I agree with the earlier answers, PMA/Ionomycin is better than PHA for activation, if you want a more physiologically appropriate activation method than you get with PMA, I would definitely recommend the anti-CD3/CD28 beads. Both Dynabeads and Miltenyi produce very effective beads for stimulation. I've used both and the big advantage of the Miltenyi ones are the size. Dynabeads are large and need to be removed before you can run them through a flow cytometer, whereas Miltenyi beads don't. If you remove the Dynabeads after 4-6 hours of culture with them, chances are most of your activated cells are still going to be attached to them, so you lose most of the cells you are interested in. Miltenyi beads run straight through the cytometer so you retain 100% of your cells.

I have delete my old account and registered a new one, so I pasted my answer here again.

"Hi Fatma

I totally agree with you and I also have experience that detecting IL-10 expression in naive T cell from EAE mice without any stimulation, but not from healthy control mice. I think stimulating drug is used just for magnifying the biological signals. Cytokines or chemokines are very hard to detect, so stimulating method is reasonable. But if we can detect the gene expression without stimulating, it is not necessary to do that.

Maybe you could isolate the naive t cells by FACS or magnetic beads, and then extract mRNA for microarray or real time PCR. As you know, real time PCR is very sensitive and could be used for quantification. If necessary, Western Blot can also be used to confirm the data.

There is another thing which should be emphasized.  It is better to use cytospin to confirm the flow cytometry data. I have tried to isolate T cells, macrophages and dendritic cells by FACS. But when I stained the selected cell by Wright Giemsa stains, I found there were some non-specific cells. If your target population is TH1 or neutrofils, it should be OK. But when you want to analyze some rare population such as Treg, you should be very careful for that. 

My colleagues prefer to magnetic beads from Myltenyi to isolate naive T cells, and confirm the efficacy by flow cytometry. This method is very reliable.

Good luck for your research!

 LI"

Hi everyone,

I would like to know whether PMA should be added in combination with Ionomycin? Will PMA alone help in stimulation?

Also what concentration of PMA/iono can be used to stimulate Tcells from WBCs isolated from blood sample?

Dear Aruna, good morning

Brefeldin A (stock solution)

· Dilute in DMSO at 100 % (10 mg/ml).

Phorbol miristil-acetate (PMA)

· Dilute in DMSO at a concentration of 50 mg/ml.

Ionomycin

· Dilute in DMSO at a concentration of 0,5 mg/ml

2 mM monensin (stock solution)

· Dilute monensin in absolute ethanol under agitation.

· Add NaOH for a final concentration of 1 mM.

CULTURE AND STIMULATION OF THE CELLS

· PBMC is obtained as usually, count the viable cells and adjust the concentration to 2 X 106 cells/ml.

· Plate in sterile 12 wells culture plates, 3 ml per well and stimulate with PMA (150 ng = 3 ml) and ionomycin (1.5 mg = 3 ml) for 4 to 18 hours, depending on the protocol, adding Brefeldin A (30 mg= 3 ml) or Monensin (1-3 mM) if you look for a protein that is secreted by the cells, to block Golgi apparatus.

Ionomycin opens the Calcium channels and Phorbol esters activate protein kinase C, therefore both are necessary to activate cells accordingly.

Hope it helps!

Many thanks to everyone that has contributed to this highly informative thread. The information are quite useful. I also recently stimulated my cells with anti CD3/Cd28 then added breferdin A as protein inhibitor before proceeding to my assay and I obtained pretty good result.

Hello, everyone! Some time ago I ran across a protocol in which brefeldin A was added 2 hours after stimulation with PMA+ionomycin. Then cells were incubated with brefeldin A for additional 2 hours and then processed for flow cytomtery. But I failed to find this protocol at present, and I am not sure if it is correct. Do you normally add brefeldin together with PMA+iono? Or give cells some time to be activated without it? Thank you in advance!

Dear Irina,

I usually add Brefeldin A after 30' and incubate cells for 4-6 hours (not overnight, to avoid over-culture effect, especially for mitogen activation). Bye

Irina, in the past I have used a combined Biolegend Cell Activation Cocktail (with Brefeldin A) (Cat: 423303) (https://www.biolegend.com/en-gb/products/cell-activation-cocktail-with-brefeldin-a-9407) that has always worked fantastically for staining intracellular cytokines, for flow cytometry.

The suggested concentration is 2 uL cocktail / 1-2 x 10^6 ml of cells; I have found this to be flexible, and worth optimising for your cell type and cytokine of interest.

All the best!

Dear Francesca and Christof, thank you for your answers! I tried to culture cells (2x10^6 ml PBMC) for 1 hour with PMA (50 ng/ml)+ionomycin (1 ug/ml). Then added 1 ul of Golgi Stop with brefeldin A from BD. And cultured cells for additional 4 hours. But the final result was unsatisfactory: I have got a lot of cells that looked like they were in apoptosis or dead cells. I have downloaded the dotplot and images of these cells from Amnis FlowSight - it is region R4 in dot plot. Are these really dead cells? Should I reduce the cell concentration or incubation time? Also these cells are from patients after a week of acute cardiovascular event. Is it possible that these cells are more sensitive to activation? Thank you!

Hi all, thanks for all your contributions above. I want to stimulate T - cells with anti CD3a/CD28 for intracellular cytokine staining, however i have monensin as a protein transport inhibitor instead of Brefeldin A, etc . When should i add monensin exactly?. After cell stimulation?. If so, how long should i exactly stimulate cells before adding monensin? and how long should i incubate stimulated cells after adding monensin?. Can some one help?