Materials

• Citrate Buffer (32 g sodium citrate, 4.2 g citric acid/1000 ml)
• Ammonium Chloride (0.85%)
• RPMI medium

Methods

1. Collect whole blood in a tube containing citrate buffer (dilute 1:8 with blood)
    
2. Centrifuge 25 minutes at 2200 rpm (1000xg), 4^o C. Use a tall and narrow tube to visualize buffy coat.
    
3. Remove the buffy coat with a glass pasteur pipette. Start at the edge of the buffy coat next to the glass - no need to remove the plasma first.
    
4. Wash cells in 10 ml of RPMI medium. Centrifuge 5 minutes at 1000 rpm (250xg), 4^oC.  Aspirate medium.
    
5. Add 5 to 10 ml of 0.85% NH₄Cl (depending on amount of blood initially collected) and resuspend cellular pellet. Let sit at room temperature for 5 min.
    
6. Centrifuge 5 minutes at 1000 rpm (250xg), 4^oC. Aspirate and wash with RPMI medium.
    
7. Cells are now ready for immunofluorescence, cell culture or RNA/DNA extraction.