ANNEXIN V staining for FLOW - (Dec/08/2011 )

hello, i was wondering if anyone had any insight into the binding buffer used for the annexin v staining for flow. i've done the staining with and without the binding buffer for total mouse bone marrow. with i get ~40% of cells apoptosing and without ~2%. i realize that annexin v + PS binding requires Ca2+ which the binding buffer provides, but is 40% really accurate????

any input is appreciated...thanks in advance for your help!

Do you use live/dead staining and lysis of red blood cells?

I use the BD Biosciences Annexin V flow staining kit, so it's coupled with PI. I do NOT lyse the red blood cells...

So you use AnnV-GFP and PI together? Do you compensate for spectral overlap?
Also, red blood cells are autofluorescent, so you would expect more false positive events in the GFP channel. Better lyse them or perform density centrifugation to compare. 40% is a bit much.