Working with murine blood to look at the population of the circulation neutrophils on the flow
need to lose RBCs and not activate the leukocyte population
We are using BD Pharm Lyse, Cat 555899, and haven't had problems with RBC lysis.
Yes, it is. We are incubating on room temperature for 14 min in dark, 5 ml of 1x lysis buffer directly added on 500 ul of whole blood or splenocyte cell suspension. We usually get >95% live cells, determined by Trypan blue counting.
ACK could make leukocytes fragile during flow cytometry. But if you wash it with HBSS-FBS usually this is not a problem. It is usual method to remove red blood cell contamination from populations before many functional analysis.
Lyse at room tempreture (usually more than 7 min). If you can read printed text through solution it's enough.
Your other question was related to Apoptosis, so if you want to analyze apoptosis in neutrophils by Annexin V - PI take in mind what membranes from lysed red blood cells could take away Annexin V and hence get you a lot of troubles (false negative results, weak signal in nucleated cells). If so, than try to use method with mitohondrial dyes (i.e. DIOC(3)6)) or membrane integrity associated methods (follow this link https://www.researchgate.net/post/Any_advice_on_Annexin_V_and_condition_without_extracellular_calcium)
Sure you will find right way!
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