A. Lysis of Mouse Spleen RBCs:
1. Harvest mouse spleen and prepare a single cell suspension.
2. Pellet the cells by centrifugation (350 x g); aspirate the supernatant.
3. Dilute the 10X Red Blood Cell Lysis Buffer to 1X working concentration with deionized water and resuspend the pellet in 5 ml of 1X Lysis Buffer.
4. Incubate on ice for 4-5 minutes with occasional shaking.
5. Stop the reaction by diluting the Lysis Buffer with 20-30 ml of 1X PBS.
6. Spin the cells (350 x g) and discard the supernatant.
7. Resuspend the pellet in the appropriate buffer (e.g., BioLegend Cell Staining Buffer Cat. No. 420201), wash 1X.
8. Count cells, adjust density, and proceed with cell staining procedures.
B. Lysis of Human Peripheral Blood RBCs:
1. Dilute the 10X RBC Lysis Buffer to 1X working concentration with deionized water. Warm the 1X solution to room temperature prior to use.
2. Add 2.0 ml of 1X RBC Lysis Buffer to each tube containing up to 100 µl of whole blood.
3. Gently vortex each tube immediately after adding the lysing solution. Incubate at room temperature, protected from light, for 10-15 minutes.
4. Centrifuge 350 x g for 5 minutes. Aspirate supernatant without disturbing pellet.
5. Resuspend the pellet in the appropriate buffer (e.g., BioLegend Cell Staining Buffer, Cat. No. 420201), wash 1X.
6. Resuspend and proceed with further procedures.