8.2 Leukocytes (Buffy Coat) Isolation Protocol

For this procedure we have used BD Pharm Lysis buffer. The below protocol was given to us by a neighboring lab (ask if you need to know the source of this protocol):

  1. Collect blood in heparinized (green cap) or EDTA (Purple cap) coated tube.
  2. Transfer 500 µl of the blood in a 50 mL conical tubes (> 500 µl).
  3. Make 1x BD Pharm lyse buffer (from 10X stock).
  4. Add 5 mL of 1x buffer .
  5. GENTLY vortex for 5 seconds.
  6. Place in dark cabinet for 10 minutes.
  7. Centrifuge 200g 5 min.
  8. Discard the supernatant.
  9. Gently re-suspend and wash the pellet with 1x PBS, 2% FBS and centrifuge at 350g for 5 min.
  10. Repeat wash step, until solution is mostly clear.
  11. Re-suspend the pellet (cells) in a small volume (1 ml) of 1x PBS, 2% FBS, and then proceed with further cleaning or DNA isolation.

8.2.1 Further Washing and Platelet Removal (Optional)

Washing the cell isolate

  1. Estimate the volume of the transferred mononuclear cells. Add at least 3 volumes (~ 1.5 ml) of balanced salt solution (PBS) to the mononuclear cells in the centrifuge tube.
  2. Suspend the cells by gently drawing them in and out of a pipette.
  3. Centrifuge 1600rpm for 20 min at 18ºC to 20°C. (Note: A centrifugation at high speed increases the mononuclear cell recovery. However, if it is important to also get rid of platelets a lower centrifugation speed is recommended (60 to 100 × g).)
  4. Remove the supernatant.
  5. Resuspend the mononuclear cells in 1.5 to 2 ml balanced salt solution.
  6. Centrifuge 1600rpm for 20 min at 18ºC to 20°C.
  7. Remove the supernatant.
  8. Resuspend the cell pellet in media appropriate for the application.
  9. Freeze immediately on dry ice.
  10. Store at -80ºC.