10x RBC Lysis Buffer (100ml)

Red Blood Cell (RBC) Lysis Buffer has been designed, formulated, and tested to ensure optimal lysis of RBCs in single cell suspensions with minimal effects on leukocytes. Nucleated RBCs are not effectively lysed with ammonium chloride. This 1x RBC Lysis Buffer is ready to use.

Components

• NH₄ Cl (1550 mMol/L)
• KHCO₃ (100 mMol/L)
• EDTA (1 mMol/L)

Application Note

Lysis of Human Peripheral Blood and bone marrow 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. For whole blood lysis, use 20 times the volume 1x RBC lysis buffer per sample volume, e.g. add 2.0 ml of 1X RBC Lysis Buffer to tube containing up to 100 μl of whole blood.
3. Gently vortex each tube immediately after adding the lysing solution. Incubate on ice or in fridge (4°C), protected from light, for 10-15 minutes.
4. Centrifuge 350 x g for 10 minutes. Discard supernatant without disturbing pellet.
5. Resuspend the pellet in the appropriate buffer (e.g. PBS or wash buffer), wash 1X.
6. Resuspend and proceed with further procedures.

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 RBC Lysis Buffer to 1X working concentration with deionized water and resuspend the pellet in 5 ml of 1X Lysis Buffer.
4. Incubate on ice or in fridge (4°C) 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. PBS (cat.) or wash buffer (cat.)), wash 1X.
8. Count cells, adjust density, and proceed with cell separation or cell staining procedures.