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):
- Collect blood in heparinized (green cap) or EDTA (Purple cap) coated tube.
- Transfer 500 µl of the blood in a 50 mL conical tubes (> 500 µl).
- Make 1x BD Pharm lyse buffer (from 10X stock).
- Add 5 mL of 1x buffer .
- GENTLY vortex for 5 seconds.
- Place in dark cabinet for 10 minutes.
- Centrifuge 200g 5 min.
- Discard the supernatant.
- Gently re-suspend and wash the pellet with 1x PBS, 2% FBS and centrifuge at 350g for 5 min.
- Repeat wash step, until solution is mostly clear.
- 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
- 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.
- Suspend the cells by gently drawing them in and out of a pipette.
- 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).)
- Remove the supernatant.
- Resuspend the mononuclear cells in 1.5 to 2 ml balanced salt solution.
- Centrifuge 1600rpm for 20 min at 18ºC to 20°C.
- Remove the supernatant.
- Resuspend the cell pellet in media appropriate for the application.
- Freeze immediately on dry ice.
- Store at -80ºC.