Red Blood Cell Lysis Using ACK Lysing Buffer

  1. Collect whole blood by venipuncture in EDTA-treated collection tubes.
  2. Pipette 1mL EDTA-treated whole blood into a tube containing 10-20 mL of ACK Lysing Buffer at room temperature.
  3. Allow the blood sample plus ACK Lysing Buffer to incubate at room temperature for 3 – 5 minutes. Lysis of the red cells should be evident during this incubation.
  4. Collect the white blood cells by centrifugation at 300 x g for 5 minutes at room temperature.
  5. Aspirate the supernatant, leaving approximately 50 uL to avoid disturbing the pellet.
  6. Gently mix the cells and the remaining fluid, then add 5 mL cold phosphate buffered saline.
  7. Mix the cells and phosphate buffered saline, and then collect the cells by centrifugation at 300 x g for 5 minutes at 2-8°C.
  8. Aspirate the supernatant and resuspend the cells in phosphate buffered saline, supplemented with carrier protein if desired, at 2-8°C.
  9. The cells are ready for further analysis.