Ammonium chloride potassium ack lysis buffer

21 days after tumor challenge, spleen, draining lymph node, and TC-1 tumors from the tumor bearing mice treated with various treatment regimens were surgically excised using sterile technique, placed in RPMI-1640 medium containing 100U/ml penicillin and 100 μg/ml streptomycin and washed with PBS. The spleen and lymph node were ground and filtered through 70-μm nylon filter container to remove undigested tissue fragments, and then 2ml of ammonium-chloride-potassium (ACK) lysis buffer (Sigma-Aldrich) was used to lyse the red blood cells. The solid tumors were then minced into 1- to 2-mm pieces and immersed in serum-free RPMI-1640 medium containing 0.05 mg/ml collagenase I, 0.05 mg/ml collagenase IV, 0.025 mg/ml hyaluronidase IV, 0.25 mg/ml DNase I, 100 U/ml penicillin, and 100 μg/mlstreptomycin and incubated at 37 °C with periodic agitation. The tumor digest was then filtered through a 70-μm nylon filter mesh to remove undigested tissue fragments. The resultant single spleen, lymph node, or tumor cell suspensions and tumor-infiltrating lymphocytes were washed twice in Hank's buffered salt solution (HBSS) (400g for 10 min), and viable cells were determined using trypan blue dye exclusion.