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Tissues and cells sample 1. Digest the fresh tissues, scatter to suspension by appropriate method. Collect mouse spleen cells to prepare a single-cell suspension. 2. Pellet the cells by centrifugation at 400-500 g at 4°C and aspirate the supernatant. 3. Add Red Blood Cell Lysis Buffer into cell pellet (3-5 times than cell pellet volume), i.e. Add 3-5 ml of Red Blood Cell Lysis Buffer into 1 ml of cell pellet. Stroke slightly and mix thoroughly, and incubate on ice for 4-5 min. During lysis, shake occasionally to promote erythrocytes lysed. (This step can be operated at room temperature or 4°C). 4. Centrifuge at 400-500 g at 4°C for 5 min, remove the red supernatant. 5. Repeat the Step 3 and 4 once if the erythrocyte lysed incompletely. Usually, very small volume of erythrocyte do not affect the follow-up detections. 6. Resuspend the pellet in PBS, HBSS, normal saline or serum-free medium and wash for 1-2 times. Centrifuge at 400-500 g at 4°C for 2-3 min, discard supernatant. The volume of wash buffer should be at least 5 times than cell pellet volume. 7. Perform a cell count after resuspending the pellet. Chongqing Biospes Co., Ltd Product Manual