I understand that to be able to measure intracellular cytokines there is a need to restimulate with PMA and Ionomycin but I don't understand how the results will be reliable after that. PMA and Ionomycin will be able to activate general different pathways and make the cell go crazy and secret different types of cytokines that may not even be relevant to your essay. If you are staining to phenotype your cells or to detect the effect of certain treatment, wouldn't this be affected by the restimulation especially if you are using a TCR targeted activation with CD3/CD28 beads?
Most protocols and vendors recommend re-stimulation and I need to use it too but I am looking for a scientifically justified reason for that!
Hello Asma El-Howati
Your questions is quite legitimate. Indeed, PMA/ION acts like a super-antigen and bypasses part of the normal activation chain of events, resulting in a super-activation. This is unphysiological and therefore a downside.
The reason people use PMA/ION before intracellular cytokine staining is to literally force the cells to produce a great amount of cytokines in a short period of time (4-5 to 24h). That is because you want to clearly see the maximum secretory potential of your cells. Also, such intense cytokine production will help you differentiate more clearly between cytokine producing populations.
However, if you apply different culture conditions or some therapeutic molecule to your cells, you will always have control samples. This helps you identify whether your culture conditions/therapeutic molecule have had any effect on your cells. That is because in the end you will stimulate both control and test cells with PMA/ION, therefore any difference between the two lots is probably caused by your culture conditions/therapeutic molecule.
Hello Asma El-Howati
Your questions is quite legitimate. Indeed, PMA/ION acts like a super-antigen and bypasses part of the normal activation chain of events, resulting in a super-activation. This is unphysiological and therefore a downside.
The reason people use PMA/ION before intracellular cytokine staining is to literally force the cells to produce a great amount of cytokines in a short period of time (4-5 to 24h). That is because you want to clearly see the maximum secretory potential of your cells. Also, such intense cytokine production will help you differentiate more clearly between cytokine producing populations.
However, if you apply different culture conditions or some therapeutic molecule to your cells, you will always have control samples. This helps you identify whether your culture conditions/therapeutic molecule have had any effect on your cells. That is because in the end you will stimulate both control and test cells with PMA/ION, therefore any difference between the two lots is probably caused by your culture conditions/therapeutic molecule.
Hello Asma El-Howati
Could you give more informations on your assay (objectives, primary vs cloned cells, etc)? In fact, PMA/Iono stimulation is not mandatory to measure intracellular cytokines by T-cells, Ag-specific stimulation is enough after short term culture if memory cells are present in the sample. Indeed, if you add PMA/Ionomycin to your antigen-stimulation wells, you won't be able to identify a specific response as (almost) all T-cells will produce cytokines depending on their differentiation TH profile (completely submerging Ag-specific rare T-cells...). We use PMA/Iono stimulation as a separate control condition, allowing to validate that the stimulation and detection steps of the Ag-specific assays were well done, as well as the capacity of sample T-cells to secrete cytokines. This could be different if you are using cloned, specific T-cells...
As an example of primary T-cells assay you can take a look in our paper detecting Sars-CoV-2-specific T-cells in ICU patients :
https://www.frontiersin.org/articles/10.3389/fimmu.2020.580250/full
PMID 33178207
Hope it helps.
Depends on your downstream applications/stimulation time. Short time of stimulation (4-6hours) might not need CD3/cd28. We had tried intracellular staining of th17 and the cytokines without Pma and iono, but didn't get a good enough signal compared to samples with stimulation.
Hi Ion Bogdan Mănescu ,
Thank you for your response, this is really great point to debate using the restimulation. I have used control cells which only had IL-2 supplement, it literally doesn't cause any stimulation but at least prevents my cells from dying within a week so I can perform my analysis with them as a negative control.
I had two groups of samples, stimulated with PMA/Iono and unstimulated, and I can see a slight difference in production between the two. surprisingly, my unstimulated show more secretion of the IFN-g & T-bet in the Th1 conditions than the stimulated with PMA/Iono. While my stimulated CD8 secreted more Granzyme than the unstimulated because Granzyme is a general marker of cytotoxicity. This may indicate that restimulation with PMA/Iono drifts the cells away from a specific physiological pathway into a more general and powerful stream of cytokines. If there is evidence that restimulation actually will potentiate the existing effect rather than drive into a general maximum secretion of everything would be great!
Hi @ Marcelo De Carvalho Bittencourt
Thank you for your response, That is great work thank you for sharing.
I am isolating Naive CD4 and CD8 from buffy coat and differentiating into Th1 and cytotoxic CD8, I am measuring IFN-g & T-bet to phenotype Th1 with Flow and Granzyme for CD8. I have used PMA/Iono as stated above in my response to Ion with great result for even the unstimulated samples. I am worried that PMA/Iono would be drifting the Th1 pathway into more general pathway such as Th0 where you can also see Tbet and IFN-g besides other cytokines not relevant to Th1. do you mean that you use it as a positive control to compare the results of your sample test.
Also, is TCR activation through CD3/CD28 enough to resemble physiological process or would you recommend antigen-specific activation in the presence of dendritic cells?
again, many thanks
Asma
Hi @ Raghumoy Ghosh ,
Thank you for your response. I had a good result with the PMA when activating something general and not as great as the unstimulated when its specific response as stated above in my other replies but most literature agrees with you that stimulation is needed I am just worried how much this has affected the specific response and wanted to be able to defend my work.
I am also interested in polarising Th17, will give it a try soon. could you please share with me tips of polarising conditions and what markers did you use to confirm phenotype. Also, do you have a paper to recommend? That would be much appreciated. Thank you in advance
Asma
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining