This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Springer Lab Protocol: Purification of human mononuclear cells and neutrophils

Purification of human mononuclear cells and neutrophils

by Chang-Duk Jun, 01/19/2000




  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


Springer Lab | See Other Protocols