Anti phospho akt thr308 rabbit mab

Spleens were harvested from 7-8 week old C57BL/6 mice and minced through a 70 µm cell strainer. After washing, the cells were resuspended in 0.165 M NH₄Cl and left undisturbed for 11 minutes on ice to lyse red blood cells. Splenocytes were enriched for T cells by depletion of B cells using magnetic anti-mouse IgG beads (ThermoFisher; cat. no. 11031). To induce ICOS expression, cells were cultured in flat-bottom 96 well plates (ThermoScientific) pre-coated with 5 µg/mL anti-CD3 (145-2C11) overnight. Cells were harvested and 5×10⁶ cells were aliquoted into tubes according to treatment and starved for 30 minutes in 100 µl PBS (containing 10 mM HEPES) at 37ºC. Cells were pre-incubated 5 minutes with anti-ICOS (7E.17G9) prior to addition of anti-CD3 (145-2C11) and anti-ICOS (C398.4A) all to a final concentration of 50 µg/mL and kept in water bath at 37ºC throughout. At 0, 5 and 15 minutes after addition of antibodies, 30µl was harvested from each treatment and added to 900 µl ice cold PBS and put on ice. Samples were lysed and analyzed by western blotting as previously described [47 (link)] using anti-Phospho-AKT (Thr308) Rabbit mAb and anti-AKT (Cell Signaling Technology)