I'm trying to set up a reproducible and reliable assay to evaluate the activity of small molecules on IL8 induced chemotaxis through CXCR1 receptor. I'm now using buffy coat isolated neutrophils in boyden chamber assay but the assay is extremely variable. I'm looking for any suggestion to improve my assay from cellular model to type of chemotaxis assay.
Dear Marica,
I would always recommend to use neutrophils freshly isolated from whole blood, not buffy coat. As you probably know, neutrophils are very sensitive and get easily activated. With cells from a buffy coat you risk getting cells that have been activated to different degrees depending on how they were handled before you got them. I have attached our paper with data on IL-8 mediated chemotaxis of freshly isolated neutrophils (and a protocol) if you want to have a look.
Thank's a lot Christina....I perfectly agree with you the only doubt I had was the amount of fresh blood that I need to get enough PMN for a chemotactic assay.
You really don't need much. 1ml of blood contains roughly 4.5-8 Mio leukocytes, and of these 50-70% are neutrophils, depending on the donor. Of course, you will lose some cells during isolation, but you should still get enough for even a very big assay. Buffy coats have a much lower percentage of granulocytes than fresh blood. I recommend to use heparin as anti-coagulant. Good luck!
Dear Christina I've read that PMN are activated by contact with glass...this will render unuseful blood samples commonly stored in glass vacutainer. Do you have experience in this?