Date: 10 October 2008

Author: TL & Rgi

Procedure:

1. Transfer 10 ml whole blood from heparin syringe (or tube) to a 14mL falcon tube and spin down at 900 x g (2300 rpm in Jouan), 10 min., RT.
2. Remove 1mL plasma and transfer to 1.8mL cryovial for later analysis. Store at -80 oC.
3. Dilute remaining blood+plasma to 30mL in DPBS, mix by pipetting,
4. Add 10 ml room temp 1.077g/mL Ficoll Hypaque solution to a 50mL Falcon tube.
5. Gently overlay diluted blood on top of Ficoll Hypaque.
6. Spin tubes 400 x g (1500 rpm in Jouan), 30 min., RT (acc=1, dcl=1, no brake).
7. Collect PBMCs from interphase of PBS/ Ficoll with a transfer pipette and transferred to new 50mL tube,  add DPBS to 40 ml.
8. Spin cells down 240 x g (1000 rpm in Jouan), 8 min., RT.
9. Decant supernatant and add 5mL  RBC Lysis Solution to pellet; incubate with mixing by inversion for 5 min. at RT
10. Dilute cells to 10mL with DPBS and spun down cells at 240 x g (1000 rpm in Jouan), 8 min., RT.
11. Decant supernatant, resuspend cells in 10mL DPBS.  Remove 125uL for cell counts, and spin the rest at 240 x g (1000 rpm in Jouan), 8 min., RT.
12. Count cells with Beckman Coulter Z1 Particle Counter, 6um particle size. 
13. Decant supernatant and resuspend cells in 10mL DPBS for final wash.  Spin down cells at 240 x g (1000 rpm in Jouan), 8 min., RT.
14. Add Lysis Buffer #5  with inhibitors (see below) to achieve 10⁸ cells/mL.
15. Transfer to 1.7mL microfuge tubes, vortex on high 15 sec., incubated on ice 5 min. repeat 2x.
16. Spin down cell debris 20,000 x g (14k rpm in Eppendforf microfuge), 20 min., 4°C.
17. Transfer supernatant to 1.0 mL cryovial and store in liquid N2.
    
    

Solutions and Materials:

• 14 ml Falcon tube (BD Falcon Ref# 352096)
• 50mL Falcon tube (BD Falcon Ref# 352073)
• 1.8 mL cryovial (Nunc # 377267)
• 1.0 mL cryovial (Nunc # 377224)
• DPBS: 1x Dulbecco's Phosphate-Buffered Saline, Invitrogen Cat.# 14190-144
• Ficoll Hypaque: Histopaque®-1077 (Sigma Cat# 10771)
• RBC Lysis Solution:  5 PRIME No.:2301310, Fisher Cat.# FP2301310.
• Lysis Buffer #5 (150 mM NaCl, 20 mM b-Glycerophosphate, 10 mM NaF, 1mM EDTA, 0.5% Triton X-100, 0.005% Tween 20, filter sterilze), add 1/100 volume of EA. inhibitor(Sigma Protease inhibitor (P8340), Sigma Phosphatase cocktail I (P2850) and Sigma Phosphatase cocktail 2 (P5726)).

Equipment:

Jouan CR3i table top refrigerated centrifuge

Beckman Coulter Z1 Particle Counter

Eppendforf refrigerated microfuge (model 5417R).