1. Make red blood cell lysis solution by combining materials below and leaving out at room temperature
4.5 mL double distilled water (room temperature)
0.5 mL red blood cell lysis solution (10x)
2. Aspirate supernatant without disturbing pellet
3. Add 1 mL of prepared red blood cell lysis solution and resuspend pellet using the least amount of force as possible
4. Add additional 2-4 mL of prepared red blood cell lysis solution depending on pellet size and presence of red blood cells
5. Vortex for 5 seconds and incubate samples for 2 minutes at room temperature
7. Centrifuge samples at 300 x g for 7 minutes
*if red blood cells still visible repeat steps 1-7*
8. Aspirate supernatant without disturbing pellet
9. Resuspend pellet in plain DMEM or RPMI 1640 media (80-500 L depending on pellet size, want to use smallest volume of media as possible while still fully resuspending pellet with minimal force)
10. Count using countess or hemocytometer (1:1 cell solution:trypan if small pellet, 1:1 cell solution:media then 1:1 diluted cell solution: trypan if large pellet)
11. While counting check for dead cells and cellular debris
If acceptable viability (>80%), proceed with sample for subsequent experiments
If unacceptable viability (<80%) and large presence of cellular debris, proceed with additional clean up