Red Blood Cell Lysis Buffer

Tissue and Cell samples

1. For tissue samples, digest and suspend using an appropriate method. For mouse spleen cells, collect and prepare to a single-cell suspension.
2. Pellet the cells by centrifugation at 400-500 × g at 4 °C. Discard the supernatant.
3. For every 1 ml of cell pellet, add 3-5 ml of Add Red Blood Cell Lysis Buffer. Mix thoroughly by gently pipetting up and down, then leave on ice for 4-5 minutes. During lysis, shake gently at room temperature or at 4 °C to encourage lysis of erythrocytes.
4. Centrifuge at 400-500 × g at 4 °C for 5 minutes. Discard the red-coloured supernatant.
5. Repeat Steps 3 and 4 if the erythrocyte lysis is incomplete. Usually, a very small volume of erythrocytes does not affect detection.
6. Resuspend the pellet in PBS, HBSS, normal saline or serum-free medium and wash 1-2 times by centrifuging at 400-500 × g at 4 °C for 2-3 minutes and discarding the supernatant. The volume of the wash buffer should be at least 5 times greater than the cell pellet volume.
7. Perform a cell count after resuspending the pellet.

Blood samples

1. Centrifuge fresh anticoagulated blood at 400-500 × g at 4 °C. Discard the supernatant.
2. For every 1 ml of cell pellet, add 6-10 ml of Add Red Blood Cell Lysis Buffer. Mix thoroughly by gently pipetting up and down, then leave on ice for 4-5 minutes. During lysis, shake gently at room temperature or at 4 °C to encourage lysis of erythrocytes.
3. Centrifuge at 400-500 × g at 4 °C for 5 minutes. Discard the red-coloured supernatant.
4. Repeat Steps 2 and 3 if the erythrocyte lysis is incomplete. Usually, a very small volume of erythrocytes does not affect detection.
5. Resuspend the pellet in PBS, HBSS, normal saline or serum-free medium and wash 1-2 times by centrifuging at 400-500 × g at 4 °C for 2-3 minutes and discarding the supernatant. The volume of the wash buffer should be at least 5 times greater than the cell pellet volume.
6. Perform a cell count after resuspending the pellet.
Note: For very small blood sample volumes, skip Step 1 and add 10 ml of Red Cell Lysis Buffer for every 1 ml of cell pellet in Step 2. For mouse blood samples, it is sufficient to lyse for 4-5 minutes. For human peripheral blood, the lysis time can be increased to 10 minutes (do not exceed 15 minutes). During lysis, shake occasionally to encourage erythrocyte lysis.