[Immunology] Human Peripheral blood's Neutrophil isolation

Milena_unipd via immuno%40net.bio.net (by milena.bordignon from studenti.unipd.it)
Tue Oct 19 09:07:47 EST 2010


  Dear Bryan Heit,
I'm Milena Bordignon from Italy (sorry for my English)!!! I've read your 
protocol on neutrophils isolation and I have some problems: I've tryed 
to isolate neutrophils from bovine whole blood to test ROS production by 
chemiluminescence. So the protocol that I use is similar to yours: I use 
Ficoll and Hypotonic lysis buffer, the procedures are similar but my 
neutrophils are often activated, after isolation, without any stimulus, 
and I don't know why...
HBSS is without Ca and Mg that can activated neutrophils, I don't use 
glass stuff, but I conserve blood at 37°C to avoid to shock cells at low 
temperature... the passage to phisiological 37°C to 4 °C do not stress 
cells???
I need some advices...
Thank's
I hope to have been clear

Milena

-- 
Milena Bordignon
PhD Student
Department of Experimental Veterinary Science
University of Padua, Italy
Tel.049-8272546



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