Isolation of peripheral blood cells FGB

Revised 8/26/96

Isolation of Peripheral Blood Cells (PBC's) .

Note: all steps performed aseptically.

1. Order blood from the Red Cross, Durham, NC.

2. Turn centrifuge to RT.

3. In hood, alcohol fresh blood bag and tubing.

4. Empty blood bag like toothpaste (~30ml) into 50ml conical tube.

5. Bring up to 48ml with HBSS (RT).

6. Add 17ml HBSS (RT) to 5-6 new tubes.

7. Aliquot blood into above tubes with 8ml each after mixing gently.

8. Add 20ml Ficoll gently from bottom so as not to disturb layer.

9. Spin at 2000 rpm/RT/30 min./brake off.

10. Suck off super. above interphase with sterile aspirator.

11. Collect ~10 ml of the interphase and concentrate 6 tubes into 4 new ones.

12. Add excess HBSS to 45ml mark and mix gently.

13. Spin at 1500 rpm/4'C/10min.

14. Suck off super, to the pellet add 10ml of RPMI 1640 +4, mix gently and collect into two tubes.

15. Spin down at 1000 rpm/4'C/10min.

16. Suck off super, add 10 ml of RPMI 1640 4+ to each tube, mix gently. If >1 tube, pool and bring up to 20ml (10ml/tube).

17. Spin at 750 rpm/4'C/10min. Aspirate, add 20ml RPMI 4+ to pellet.

18. Repeat one more time.

19. Combine pellets in 20-30ml of RPMI 4+ and resuspend well.

20. No clumps here. Gently aliquot suspended cells 5ml each to tissue culture grade petri dishes, spread well and incubate at 37'C for 1-1.5 hours to adhere.

21. Once adhered, mix well by swirling and zig zag, collect and save floating cells T cells, NK cells, B cells etc. in a 50ml conical tube (1 or 2 tubes), one or two at a time. Replace lids ASAP!

22. Then add 5ml of 37'C RPMI 4+ to the side of dishes to collect extra floating cells, swirl and collect into same tube.

23. Wash 2X, again collecting the solution .

24. To adhered cells add 10ml warm RPMI 1640 4+ and leave for 1 week in 37'C incubator to differentiate.

25. In 5-7 days see isolation of monocytes protocol and follow.