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
