Hi, guys. I want to use MACS-sorted mouse CD4+ T cells to test the effect of an drug and some directors asked me to culture T cells without IL-2/CD3/28 to exclude the effect of these stimulation. However, I cultured it and find that cells under 16h culture broken into pieces when I am using flow cytometry for analysis. May I know how long (how many hours) can I keep MACS-sorted CD4 T cells without IL-2? I found if the culture time is too long they will die, such as 24h. But how long is enough without killing them? May I know your experience in T cells culture?
We used to culture them with antigen presenting cells, here we did not add IL-2 for the first 24h. But they need to grow quite dense ( low volume) , if they are to separated they don´t get enough growth factors ect.. does your RPMI contain glucose and pyruvate?
Hi Shuoyang.... Since you are culturing sorted T cells, you will definitely require IL-2 for long time cultures. I have worked on isolated human CD4+ T cells and overnight incubation didn't affect viability. However, health of cells at the time of sorting and time taken for sorting i.e time from isolation of PBMCs from blood/natural microenvironment to getting sorted cells will have a significant impact on cell health and viability.
Another approach, if the time to keep in such medium is unavoidable, you can consider to use suboptimal IL-2 (25-50 IU/ml in place of normal 200 IU/ml) without anti CD3/CD28. I hope that will help you.
Good luck
Your T cells will definitely need some stimulus to survive further than 12h. If they do not receive any proliferative stimulus of any kind, they will start undergoing apoptosis after a few hours. Anti-CD3/28 and/or IL-2 are the typical stimuli. You could try IL-7, which does into induce such proliferative responses but keeps them alive. It all depends on what you are trying to answer. The people who asked you to do that without stimulation are probably not familiar at all with these kind of assays.
As I read this the CD4+ are MACS-separated, which is a relatively quick protocol, and purity will NOT be 96-98%..
For HUMAN CD4 I rest them overnight to even 2 days sometimes in X vivo after MACs sorting before using for short term assay. In a FACs course we did a comparative analysis on expression of typical markers in several human blood cells T cells were one of the most stable in terms of those marker expression. I am unsure about mouse cells though. Plus it is always advised to rest the cells atleast 6 hours after MACs sorting since sorting is itself affects the cells.
In my experience negative CD4+ T cell isolation using MACS works well; you just have to make sure your reagents are good and the sped of the protocol isn't really an issue. Having said that IL-7 is good for sustaining cells in cultures for prolonged periods but should be added regularly into the culture. The concentration does define the effect; low doses help in cell survival while high doses can cause them to proliferate. Again, your experimental design will depend on the experimental question being asked. Good Luck!
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