Anti mhci

Single-cell suspensions from individual tumors and spleens were prepared to examine their resident cell types and cell surface markers. Red blood cells were depleted with ACK lysing buffer. Cells were washed twice in PBS and counted on a hemocytometer. Cells were stained with primary antibodies or appropriate isotype controls in PBS + 1% BSA (FACS buffer) for 30 min on ice, followed by 2 washes with FACS buffer. For gp100, cells were stained with an unconjugated primary antibody, washed twice with FACS buffer, and incubated with the secondary antibody for 30 min, followed by 2 washes. Flow cytometry was performed by a NovoCyte flow cytometer and analyzed by NovoExpress software (ACEA Biosciences). For analysis, a typical forward-side scatter gate was set to exclude dead cells and aggregates. Antibodies were from Life Technologies, unless otherwise noted. These antibodies included anti-CD8 (clone, 536.7), anti-CD4 (Gk1.5), anti-CD3 (145–2C11), anti-PD-1 (J43), anti-MHC-I (AF6–88.5.5.3), anti-PD-L1 (MIH5), and antimelanoma gp100 (EP4863(2), Abcam), with the secondary antibody of goat antirabbit IgG (Alexa Fluor 488). Rat IgG2a, k (RTK2758), rat IgG2b, k (RTK4530), Armenian hamster IgG (HTK888), and mouse IgG2a, k (eBM2a) were used as isotype controls.