Hello,
I am trying to stimulate OT-1 cells in vitro and but have had a hard time getting a good IFN-g production as measured by FACS.
Here is my protocol:
I mix at different effector to stimulator ratios (1:5, 1:1, 5:1) irradiated Act-OVA spleenocytes (serving as stimulators) and naive OT-1 spleenocytes (enriched using a kit) for two days. After two days, activation is great, as observed by the increase in CD69 expression levels (94-98% CD69+) and change in morphology.
After 2 days, I spin down the cells and resuspend them in fresh media and "rest" them for an additional of 3 days after the initial priming. At day 5, I restimulate the cells with PMA/iono (6hr) + BFA (4hr) and measure IFN-g production.
I do no get a GOOD interferon-g production, why ? could anyone help me please ??
I change media every other day when color turns yellowish.
Media contains: RPMI, 10% FBS, antibiotics, 2ME, 50U IL-2.
Thank you very much !
Five days after primary stimulation seems short. It could be possible that, even though you're seeing proliferation and CD69 upregulation, your cells aren't fully differentiated effectors yet. When I work with OT-I cells, I usually do my experiments between day 7-9 after primary stim (however, I do cytotoxicity assays and I've never looked at IFNg signal from my cells). Colleagues of mine who do IFNg ELISPOTs often do the assay as late as two weeks after initial stim and are able to detect the IFNg. Perhaps holding off on the IFNg measurement might enable you to find a signal?
Also, what is your resting media for the 3-day rest period? Generally, it's a good idea to add IL-7 to the media during the resting stage (especially if you've decreased the IL-2 dosage for this step)
Ok, Thank you, I was afraid to go for a longer period than that, I was told primary mouse T cells are very sensitive, and that I could get a lot of cell death in culture.
For the culture media, I use the same media, RPMI, 10% FBS, P/S, 2ME, IL-2.
I add 50U of IL-2 to my media and keep it at 4d. I add an additional 20-30 U before incubating at 37deg.
Thank you very much for your helpful advice.
Just make sure your stimulated splenocytes don't go over 3 million/mL or they will start to die quickly. Usually this means passaging them back down to 1 million/mL on days 5, 7, 9, 11, 14.
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