Hi everyone,
I'm going to generate CAR-T cells starting with mouse CD3+ T cells. I isolated mouse CD3+ T cells using Miltenyi Pan T cell isolation kit. I seeded cells 1millon/well in 24 well plate precoated with 1ug/ml antiCD3 and 2ug/ml antiCD28. Cells were culture in the presence of 50IU/ml rhIL-2. However, only 20% of the cells were alive after 48h culture. Not a lot of cells became bigger, and I didn't see clusters forming. My question is that is it because of not sufficient TCR stimulation or AICD? I'm using RPMI supplement with PS, Glutamax, sodium pyruvate, 10% FBS, b-Me. Thank you in advance!
You can prepare 500 mL of T cell culture medium by dissolving 5 mL of 200 mM L-glutamine (final concentration 2 mM), 5 mL of penicillin-streptomycin, 1 mL of 50 mM 2-mercaptoethanol (final concentration 0. 1 mM), 1 mL 100 mM solution of sodium pyruvate (final concentration 0.2 mM) and 50 mL heat-inactivated fetal bovine serum (final concentration 10 %) in 438 mL RPMI 1640 and filter the culture medium using a sterile GP vacuum filtration system. Maintain at 4°C for up to 1 month. Than may be you will get may be more quantity T cells I HOPE .
Hi Jian,
Keep the anti-CD3 plate-bound at 1ug/ml and try using soluble anti-CD28 instead of plate-bound. You can also increase the concentration of anti-CD28 up to 5ug/ml.
Check this protocol:
https://www.thermofisher.com/uk/en/home/life-science/cell-analysis/cell-analysis-learning-center/immunology-at-work/immunology-protocols/t-cell-activation-anti-cd3-anti-cd28.html
I hope this helps.