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Springer Lab Protocol: Purification of human mononuclear cells and neutrophils

Purification of human mononuclear cells and neutrophils

by Chang-Duk Jun, 01/19/2000

Purpose

Materials

Procedure

  1. Draw venous blood into 60 ml syringe with 10ml 6% dextran + 7 ml citrate/citric acid. Fill to 60 ml total (blood 43 ml).
  2. Mix and sediment 30 min at RT (white blood cells now with serum).
  3. Transfer serum/white blood cells to 50 ml tube, underlay 12 ml RT Histopaque 1077.
  4. Spin 2500 rpm/30min at RT.
To take mononuclear cells:
  1. Take off serum above band. (See attached figure, in preparation)
  2. Take mononuclear band + some Ficoll into two 15ml tubes.
  3. Add cold media A up to 15ml.
  4. Spin at 1800 rpm 4oC for 5min.
  5. Pool pellet into one 15ml tube and add cold media A.
  6. Wash cells three times (1400 rpm, 4oC, for 5 min) to remove platelets.
  7. Add media B and count cells.
To take neutrophils:
  1. Remove mononuclear band and serum with suction. (See attached figure, in preparation)
  2. Wash in PBS (or media A) to remove Histopaque (spin 1800 rpm/5 min at 4oC)
  3. Remove PBS (or media A) , add 18 ml cold H2O to lyse RBC, and add 2 ml 10x PBS.
  4. Spin 1400 rpm/5min at 4oC.
  5. Wash as needed, resuspend, and count cells.

Expected Results

References


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