Hi, I dont know about the murine RBC lysis but for the human blood we use bufered ammonium chloride solution. hope these suggestions help. 1. the blood has to be at RT at 4 the membrane is stabilised and so lysis will not be complete. 2. we take about 2 ml blood adn dilute it to 50ml with 1x buffer. 3. mix the two gently adn then incubate at 37 for 10 min. the composition of the buffer is- nh4cl 32.08gm nah2co3 3.36 gm edta na2 1.41gm make up to 400ml with milliQ water. filtersterilize if required. this is 10x solution. can be stored at 4 for 6 months. make up to 1x immediately before using. 4. check the viability of the cells with trypan blue ater the spins. Generally we go for 1000 to 1200 rpm on a bench top clinical centrifuge. also u can try underlaying with fcs and then spinning, hope these help Regards Rana. "Haugan, Anita" <anita.haugan@folkehelsa.no> wrote: To all of you who use ammonium chloride lysing of RBC in whole blood samples from mice for surface markers: I need some advice concerning lysing of murine RBC. I have just started to work with whole blood from BALB/c mice, and are trying to lyse the RBC with a NH4Cl-solution. My problem is that my samples aren't completely lysed, and one can see that the pellets are redish in some cases. My qustions are: What is the optimal g-value for centrifugation of the samples to get maximum lysis? We use 1200rpm (270g) for 10 min. Is there anything else we should be aware of in using NH4Cl lysing - both concerning the lysing method and analyzing the samples on the flowcytometer? So if anyone have some answers/advices or best of all a detailed protocol of how to lyse RBC in murine whole blood they could send me, I will be extremly happy! Thanks in advance! Anita Haugan Department of Vaccinology National Institute of Public Health, Norway ____________________________________________________________________ Get free email and a permanent address at http://www.netaddress.com/?N=1
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