First, we coated 96 well TC-treated plates with 5ug/ml anti-CD3 antibody (17A2, biolegend)
Then, we added Naive CD4+ CD25- CD45RB high T cells (500000) in each well after sorting through flow cytometry. Next, we used anti-CD28 antibody (soluble 2ug/ml, biolegend). Again, different cytokines such as 5ng/ml TGF-β, 20ng/ml IL-6, 10ug/ml anti-IL-4, 10ug/ml anti-IFN-gamma were added in each well. After 5 days, when we detecetd TH17 cells after, we found low ratio of IL17+ cells (1%-2%) and most of cells were dead (>80%). This is the main problem. Thats why we want to know which can cause this problem.
Dear Ajmeri,
I would suggest to add a Th0 control, without cytokines, to check the activation. It seems to me that the anti CD3/28 stimulation did not work, and then you can not get Th17. Especially, in my hands, Th17 were pretty resistant to strong stimulations, and if the stimulation worked, you would have alive cells.
Suggestions:
- important: use different plates: non-treated, flat bottom plates. The usual treated plates make it difficult for anti CD3 to stick
- which time-point are you using for read-out? It is important to remove stimulation at day 3 and replate the cells on new plates without antiCD3/28, if not they will die
- I recommend using a medium with high calcium and glutamine like IMDM to differentiate Th17 instead of RPMI
- you can consider comparing your activation protocol with coating both anti CD3 and anti-CD28 together on the plate (I was using 1ug/mL of each coated 3 hours in PBS) and not using anti CD28 soluble. Of note, your soluble antiCD28 could be higher (5 ug/mL). In general, you can use ~1 ug/mL for coating or 5 ug/mL for soluble.
- adding IL1b as well could improve differentiation
- you can consider to add anti IL2, you will have more IL17+ cells but possibly less alive cells,
- of note, there will be major differences between mice and human cells. What I told you above works with murine cells, I am not sure this will work equally with human cells, and also check if the cytokines match the species.
But first, simple activation with T cells on the coated plates should work well and cells should be alive at day 3, before you can test Th17 protocol
Best
Philippe August Robert Thank you sir for your kind response and suggestion
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