Neutrophil isolation - Isolation of PBMC and neutrophils from blood (Mar/22/2010 )

Hi,

I was wondering does anyone know if it possible to isolate neutrophils and PBMCs simultaneously from whole blood? So far I have only been isolating PBMCs using Ficoll.

Thanks.

bourkenm on Mar 22 2010, 06:40 PM said:

PMN Neutrophil Cell Isolation

A easy procedure reported in article “Zagami F.:(1993) Phagocitic index: A valid method of phagocytosis study. Agg. Med. & Chir. Vol. 11, 5, 469-478, in which I showed in material and methods the experimental way.

Cellular yield: a discontinuosus gradient of Percoll (Pharmacia) is used, starting from Percoll done isotonic dissolving in it 0.15 M NaCl (solution referred at 100%). One volume of 1:2 whole blood (EDTA-K3) diluted in NaCl 0.9%, is poured in silicone glass tube. Using a syringe, connected with a 19 G Luer needle, pour below the level of the blood, one volume of 50% isotonic Percoll solution diluted in NaCl 0.9%. With the same identical procedure one volume of isotonic Percoll solution at 69% is poured below the 50% Percoll solution. Centrifuge tube at 350 x g for 20 min. The densities of the various isotonic Percoll solutions were determined using graduated density (r) beads (Pharmacia), and diluting each vial, from 1 to 9, with 1 ml of PBS. 20 µl of each contents were mixed together and placed at one volume of NaCl 0.9%. The specimen has been processed in the same identical way as the experimental of the sample. After centrifugation, the granulocytes band was removed with a Pasteur pipette and subjected at two washing with PBS, each followed by a centrifugation at 400 x g for 10 minutes. If erythrocytes contamination is present, eliminate it through osmotic lysis, resuspending pellet with one volume of 0.9% NaCl and add three volumes of distilled water, and delicately mixing for 30 seconds. At the end of time, add one volume of 3.5% NaCl in order to stop the reaction. After centrifugation at 400 x g the suspension is subjects to once wash with PBS, resuspended pellet in PBS and the recuperated cells are analyzed.