Purification of human mononuclear cells and neutrophilsby Chang-Duk Jun, 01/19/2000 Purpose Materials

• 10ml 6% dextran + 7ml citrate/citric acid
  • Dextran: T500 --> 6g+100ml PBS
  • Citrate solution: 25g Na Citrate + 8g citric acid + 500 ml PBS
• 43 ml blood
• 12 ml RT Histopaque 1077
• 18 ml cold H2 O
• 2 ml 10x PBS
• M199 for HUVECs: 1L powder pocket M199 + 35g NaHCO3 + 25mM Hepes + 5 mM glutamine + 50 ug/ml Gentamycin.
• 1M Hepes: 119.1 g Hepes + 500 ml dH2 O.
• Media A: 1X HBSS (10X:50ml) + 10 mM Hepes (1M: 5 ml) + 5 mM EDTA (0.5M: 5ml) + 2.5 % FBS (12.5 ml) in 500 ml.
• Media B: RPMI1640 470 ml + Gent (1000X) 0.5 ml + Glutamine (100X) 5 ml + 5% FBS (25 ml)

Procedure Draw venous blood into 60 ml syringe with 10ml 6% dextran + 7 ml citrate/citric acid. Fill to 60 ml total (blood 43 ml). Mix and sediment 30 min at RT (white blood cells now with serum). Transfer serum/white blood cells to 50 ml tube, underlay 12 ml RT Histopaque 1077. Spin 2500 rpm/30min at RT. To take mononuclear cells: Take off serum above band. (See attached figure, in preparation) Take mononuclear band + some Ficoll into two 15ml tubes. Add cold media A up to 15ml. Spin at 1800 rpm 4o C for 5min. Pool pellet into one 15ml tube and add cold media A. Wash cells three times (1400 rpm, 4o C, for 5 min) to remove platelets. Add media B and count cells. To take neutrophils: Remove mononuclear band and serum with suction. (See attached figure, in preparation) Wash in PBS (or media A) to remove Histopaque (spin 1800 rpm/5 min at 4o C) Remove PBS (or media A) , add 18 ml cold H2 O to lyse RBC, and add 2 ml 10x PBS. Spin 1400 rpm/5min at 4o C. Wash as needed, resuspend, and count cells.