Neutrophil phagocytosis

[Corrado Cilio]

Mike Collier wrote:
> 
> A colleague is about to undertake a project using flow cytometry to
> investigate Neutrophil phagocytosis. Does anyone have any experience /
> methods in this field using whole blood
> --
> Mike Collier       mike at mcollier.demon.co.uk
> 
> Chief MLSO
> Haematology laboratory
> Princess Margaret Hospital
> SWINDON
> WILTS
> ENGLAND
> 
> Phone 01793 426340
> FAX   01793 426827 
Dear mike,

I have done some work on human neutrophils, in whole blood, looking for 
phagocytosis and metabolic burst by FACS analysis. It was working find. I 
had a nice protocol from Sandra Perticarari (Trieste, Italy) (see 
references below). The method take advantage of hydrethidine (HE) and 
FITC-labeled bacteria (Staph or Candida) using 100 ul of whole blood. You 
can incubate the samples with PMA or with zymosan plus HE and/or bacteria 
and look for oxidative burst activation (hydroethine --ox--> Ethidium 
Bromide --> fluorscence emission on FL3) and phagocytosis (FITC on FL1). I 
did also staining for surface markers on monocytes (i.e. PE-conjugated 
anti-CD14 on FL2) in a three-color flow cytometric analysis.
If you want I can give you more details on the protocol, just let me know 
by e-mail.
I hope it will help.

REFERENCES
Prodan et al. Haematologica 1995, 80(3):212-8
Cinco et al. FEMS-Microbiol-Lett. 1994, 122(1-2):187-93
Perticarari et al. J.Immunol.Methods 1994, 170(1):117-24
Perticarari et al. Cytometry 1991, 12(7): 687-93

Good luck 


Corrado Cilio, M.D.
Dept. of Cell and Mol Biol
Univ of Umeå, SWEDEN
FAX +46-90-771420
Phone +46-90-176712-13
e-mail: Corrado.Cilio at cmb.umu.se