neutrophils

Labo Longziekten overveld at uia.ua.ac.be
Tue Apr 29 04:28:58 EST 1997


Newsgroups: bionet.immunology
Subject: neutrophils and PERCOLL

Isolating neutrophils should be very simple.

In our lab we use either Percoll or Ficoll-Hypaque to separate different type of cells.
This is our Percoll protocoll:

Prepare Percoll 1.100 g/ml first
Percoll (1.129 g/ml) 708.5 ml   or  (1.130 g/ml)  703.9 ml
conc. PBS*            57.5 ml                      57.1 ml
PBS*                  34.0 ml                      39.0 ml
GPO*                 100.0 ml                     100.0 ml
TNC*                 100.0 ml                     100.0 ml

Check the density (1.100 g/ml), pH (7.3) and osmolality (290 mOsm/kg H2O).

*concentrated PBS: 90 g/L NaCl, 13.8 g/L NaH2PO4.H2O; no pH adjustment.
*PBS: 8.2 g/L NaCl, 3.1 g/L Na2HPO4.12H2O, 0.2 g/L NaH2PO4.H2O; pH 7.4 at 20°C.
*GPO: pasteurised plasma-protein solution (CLB, Amsterdam); =40 g/L protein, 4 mmol/L sodiumcaprylate and 130-160 mmol/L sodium.
*TNC: 38 g/L tri-sodiumcitrate, 11 hydrate.

Using the formula y=0.000909x+1.0091 in which y=density and x=ml Percoll 1.100 g/ml, you can prepare every density by diluting Percoll d=1.100 g/ml (x ml) with PBS++ (80% PBS, 10% GPO, 10% TNC) up to total volume of 100 ml.

For Percoll with d=1.077 g/ml, which is used for neutrophil separation, you should mix 74.7 ml Percoll 1.100 g/ml with 25.3 ml PBS++.

This should work.

Frans J. van Overveld, Ph.D.
Dept. of Respiratory Medicine
Univ. of Antwerp



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