Hi,
I have tried culturing CD4 cells (isolated by Stemcell technologies) with plate-bound anti-CD3 (2 microgram/mL) with sterile PBS. Aspirating the PBS. Adding 200K cells/ well along with a stimulation cocktail of anti-CD28 (2microgram/mL) and IL-2 (200 ng/mL). I have not seen any kind of proliferation of the CD4 cells. Please suggest what might be going wrong in my protocol.
Hi,
for CD4 cells you don't really need to add IL2, as the cells will produce their own cytokines.
What medium are you using? Do you add 2-Me? I saw that it helps a lot to keep cells viable during proliferation. Also you can try increasing you anti-CD3 concentration at different concentrations.
how do you know that your cells are not proliferating? Do you use a cell counter? Does the number of viable live cells change?
Regards,
Simon
Thank you Simon for the reply. Yes, I tried thymidine assay to see the proliferation. I tried culturing the cells with and without IL2, to see the effect. I use RPMI complete medium without 2-ME. Maybe I should add it and see how it goes. I will also try to add different higher concentrations of aCD3 to see it the cells proliferate better.
Problem with CD4 proliferation using plate-bound anti-CD3? - ResearchGate. Available from: https://www.researchgate.net/post/Problem_with_CD4_proliferation_using_plate-bound_anti-CD3#57a49bd996b7e463331b6171 [accessed Aug 5, 2016].
Hello,
How long did you stimulate them and in which well plate did you seed them?
Hi Amarendra, I used the 96-well flat bottom plate, and did the stimulation for 72 hours.
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