Performing FACS analysis on NHP PBMCs cultured ex vivo on anti-CD3/anti-CD28 coated plates for 96 hrs. and then fixed and frozen OR treated with PMA/Ionomycin for 6 hrs prior to fixation and freezing. Seeing differential cytokine expression and wondering whether the cultured lymphocytes or the PMA/Ionomycin-treated, cultured lymphocytes are a better representation of the animals' immunophenotype.
I've done similar analysis both ways, CD3 and CD28 for a few days followed by PMA/Ionomycin treatment. You can see differential cytokine expression both ways but in my hands the PMA/Ionomycin makes the difference more dramatic, so I tend to stick with that. We've published with PMA/Ionomycin stimulation so it's accepted in the field as representative of PBMC cytokine phenotype.
anti-CD3/Cd28 activate naive T cells and later if u treat them with PMA/ionomycis ur asking these cells to secret cytokines/chemokines. This method is good if ur asking a question of what cytokines they secret and looking for ICS.
CD3/CD28 stimulation is definitively the one more physiological. As membrane permeable specific activator of the protein kinase c (PKC) and hence of the NF-kappa B PMA causes an extremely wide range of effects in cells. Combination of PMA with ionomycin is just increasing this effect. Of course one can see strong cellular reactions e.g. T-cell proliferation, expression of early and late activation markers (CD69 and CD25, respectively) and release of cytokines but they represent artificial reactions (good as positive controls). Thus I would recommend the combination of CD3/CD28 because this is accepted to represent the "physiological" T-lymphocyte reaction.
I fully agree with Bernhard. Anti-CD3 plus anti-CD28 is close to physiological, maximalizing the effect of 'natural' costimulation by CD28 ligation on available APCs (in PBMC, mostly the monocytes). Also consider, that when you stimulate initially with anti-CD3 plus anti-CD28 combination, you trigger the RESTING lymphocytes, while when you then add PMA/Iono after another 96 hours in culture, you "superstimulate" the cells that are already cycling, possibly maximalizing the available responses or, if you overload, just anergizing or even killing some lymphocytes . Have you looked at the apoptosis levels before and after PMA/Iono stimulation in your setup?
It depends on what animal immunophenotype profile do you want to analyze. In the case of one hyperstimulated, I think it would be better the stimulation with anti-CD3 plus anti-CD28 and then with PMA/ionomycin OR if you want to analyze better more normal conditions, a stimulation with anti-CD3 plus anti-CD28 is enough.
In answer to Jacek's question, the cells I am working with were counted by Trypan staining after anti-CD3/anti-CD28 stimulation or after anti-CD3/anti-CD28 stimulation and PMA/Ionomycin staining. They were then fixed and frozen for later analysis. So no apoptotic markers have been used in the analysis.
OK Matthew, so what were your viabiities by Trypan? I a still trying to get at your problem. Did you try to stimulate your cells with just PMA/Iono? How did the results compare to the other protocols?
Average recovery from culture was approx. 5-7 x 10^6 cells per culture well.
The core issue I'm having is that for many of the time-points I'm analyzing by flow, PMA/Iono stimulation does not appear to have stimulated cytokine production - I'm leaning towards over-stimulation/anergy as the reason for poor responses.
Where I do see responses following PMA/Iono stimulation, I see lower responses than those following anti-CD3/CD28 culture alone.
Moreover, responses go down in control and treatment groups with PMA/Iono whereas vehicle-treated cells show increased responses following PMA/Iono.
So my quandary is which set of stimulations best represents the immunophenotype in vivo?
I'd guess you should go for anti-CD3 stimulation of the total PBMC (as the closest to physiological) and for anti-CD3 plus anti-CD28 for isolated T cells (or PBMC, if you want the maximal available response). RE cytokine expression: do you seek/quantify the cytokines from medium (i.e. secrested) or intracellular? If the latter, what do you use to stop secretion? Brefeldin? Monensin? Golgi-stop?
We took supernatant from cultures to measure secrete cytokines via Bioplex and/or ELISA. For flow, looking at intracellular cytokines. I've used BD GolgiPlug (Brefeldin A) with success. Depending on the cytokine of interest, thoug, monesin can be as/more effective.
OK. Then, did you look at cytokine secretion profile over time, or only after 96 hours in culture? For PMA/Iono just a few (4-18) hours might be enough; physiological stimulation requires more time, but you should be able to see some accumulation or secretion after a day.
Everything up to fixing the cells was performed prior to my joining this particular lab. I've only carried out the flow cytometry on fixed, frozen cells from these two stimulations. Supernatants were collected - but I don't know the particulars and they have yet to be analyzed.
Well ok. Sounds strange but I understand the material was precious and your boss wanted to collect as much as possible for future research. So, your approaches are limited - you can only do flow and then try interpreting the results...
A second thought: if staining your thawed cells for flow, try adding some viability staining to exclude dead cells from the picture...
Michal, you right you can, but the question is what is the purpose of the experiment? To get the 'close to physiological' response, or the maximal one available for this specifoc sample? In the latter case you would stimulate in the presence of exogenous IL2 and possibly other cytokines, or max the response using PMA+Iono...
- Badges
- Science method