Hi fellow immunologist. I am having some viability issues with murine CD8 culture that I am looking for help with. Here are my general steps.
Negative selection of mouse splenocytes using the miltenyi CD8 isolation kit. Acquiring 4-12 million CD8s per spleen with approx 100% viability.
Either
(a) seed cells in wells precoated CD3/28 or another TCR engager (standard 2 hour coating protocol, using 20-50nM concentration) or
(b) treat CD8s with soluble TCR engagers (including an anti-hamster antibody for anti-CD3/28 to support crosslinking). In each condition, I have unstimulated and IL2 controls.
- 24h later, analyze cells.
My issue is that my viability is generally poor upon viability analysis after 24h culture, between 20-50% following this 24 hour incubation, across all treatments (with and without TCR engager, with IL2 or not). I have altered seeding density (1-2.5 million per mL), used flat-bottomed or round-bottom culture (the latter only with soluble engagers), and currently use the following media formulation: RPMI with L-glut, 10% FBS, 1% pen-strep, 25uM 2-ME.
Will any of you fine folk share your thoughts and experiences?