you have to activated the cells? You can use a specific antigen you are using or a non-specific stimulator such as a lectin (PHA; phytohemagglutinin). You can also coat your plate/flask with anti-CD3/CD28 antibodies which mimics signals from APCs and activate your T-cells.

Hello Thomas,

I'm sorry, I forgot to mention above that I stimulate the cells with the CD3/CD28 T-cell activator from StemCell. I will edit my inital question.

After thawing BM-MNCs, wash well to remove DMSO and add DNase I to prevent clumps. Let cells rest 12–18 h in medium with low IL-2 before CD3/CD28 activation, because too-early or strong activation often kills them. Use a lower IL-2 dose (50–100 IU/ml instead of 200) and consider adding IL-15 or keeping IL-7 for survival. Human AB serum works better than FCS, or you can use serum-free X-Vivo. Keep cell density around 0.5–1×10^6/ml, change medium after 24 h to clear debris, and handle cells gently with low-speed spins and warm media. If viability still drops, check apoptosis or mycoplasma, and try ROCK inhibitor (Y-27632) to reduce cell death

Hello Muhammad,

thanks a lot for your helpful input! We will definitely do another IL-2 titration.

Do you add IL-15 instead of IL-2, or in addition to it?