I am culturing CD4+ T cells isolated from CRISPR-Cas9 knockin mice (Platt et al. Cell 2014, 1559:440). Initial activation on anti-CD3/anti-CD28 coated wells for 2 days seems normal and cells get activated (increase in size on FSC/SSC plot). However, after day 3 those cells do not proliferate well and start dying, irrespectively whether they are left on anti-CD3/anti-CD28 coated wells or moved on non-coated wells with addition of IL-2. Does someone have advice how to keep these CD4+ T cells in culture?
I think that the test and result of your experiment can be perform by bioinformatic methods before done. Of course this is a proposal because the conditions of any experiments can affect on results.
I am doing the same experiment as Berislav, but on human CD4+ T cells. I have the similar question as Berislav: how to maitain the CD4+ T cells healthy in culture?Jia Liu, how long your T-cells can live after the activation of the CD3/CD28 activation beads from Thermo Fisher? Thanks.
In my experience either something is wrong with the culture conditions and some essential nutrient or cytokine or stimuly is missing (try different culture conditions ; enough antiCD3 and a Cd28 or too much; different plates? adding irratidtes APC etc) or something is wrong with the T cells themselves because normal CD4 T cells should stay alive under these conditions and can be kept in culture for some time, when you split them, rest them, restimulate and add new culture medium plus Il-2 as needed. Maybe these particular T cells need something additional to keep them alive. Good Luck!!
Hi Klaus, thanks a lot for the answer! We have a protocol to get the CD4+ T cells activated and proliferating (as you mentioned exchanging rounds of aCD3/aCD28 (re)stimulation with phases in medium+IL-2 for proliferation). We observed initially some differences between CD4+ T cells coming from B6 wild-type mice and from CRISPR-Cas9 knockin mice and I am wondering is it coming due to Cas9 expression. Will do some more experiments to check this issue again, but I am wondering did anyone else observed the same problem with CRISPR-Cas9 CD4 T cells?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology