I have been using an MLR protocol that hasn't turned up any results as far as cell proliferation over the period of 4-5 days. I am using cell strains from Balb/c and B6 mice as stimulators and responders. eFluor670 is being used as a proliferation dye. ConA is being used as a positive control in the concentration of 1mg/mL. Any working protocol with specifics (ex. composition of media) would be extremely helpful to continue with my research.
It should be easy but it depends on your conditions. I have not run this lately but when I was doing it all the time I used RPMI 1640 medium and 10% fetal calf serum. I also added 2 mercaptoethanol but I'm not sure how necessary that is.
The stimulator spleen cells I would add 500,000 per well of a flat bottom 96 well plate and the responder T cells at 250,000 per well. Con A at 1 mg/mL seems like way too much. 1 ug/mL should be plenty but ideally you could run a concentration curve to find the optimum. Just plate your spleen cells at 250,000 per well and add ConA at between 0.1 and 10 ug/mL in half log intervals (roughly 1:3 dilution series).
If you are reading out by flow cytometry using reduction of the fluorescent dye (sounds like you are) be sure you don't label your responders too much. It can reduce function at a high enough level.
We are measuring proliferation on day 7 in the human MLR this way. Day 4 or 5 might not be optimum. Try different days and you will find the sweet spot. Somewhere between day 5 and day 7.
Good luck!
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