1. 2 drops blood into 10mL of Gey’s Final Solution in a 15mL Falcon tube.
2. Allow to lyse at least 20-30 minutes on ice.
3. Centrifuge the tubes @ 1250 rpm for 10 minutes.
4. Dump the supernatant.
5. Put 2mL of 4% BSA in PBS (calcium and magnesium free, pH 7.4) into tubes.
6. Spin tubes @ 1500 rpm for 5 minutes.
7. Dump the supernatant.
8. Re-suspend the pellet in the roughly 300ul of BSA left over.
9. Transfer 100ul aliquots of the suspension to wells in a culture plate.
10. Centrifuge @ 1500 rpm for 5 minutes.
11. Dump the supernatant.
12. Stain with 100ul aliquots of R73.
13. Leave to sit for 10 minutes in the dark.
14. Centrifuge @ 1500 rpm for 5 minutes.
15. Dump the supernatant.
16. Add 100ul BSA to each sample well.
17. Centrifuge @1500 rpm for 5 minutes.
18. Dump the supernatant.
19. Re-suspend each sample with 200ul of PBS in 5mL polystyrene tubes for FACS analysis.
19. Add 200ul 1% Paraformaldehyde to each sample well (note that the current stock of
para is at 4%, so it must be diluted).
20. Incubate at room temperature for 20 min. in the dark.
21. Centrifuge @1500 rpm for 5 minutes.
22. Dump the supernatant.
23. Re-suspend each sample with 200ul of PBS in 5mL polystyrene tubes for FACS analysis.
24. Keep samples at 4° in the dark until analyzed.
1. R73 1:200 PE
CD8 1:200 FITC
CD4 1:150 CYCrm