I would like to ask you for your help with some problem regarding the analysis of murine (mouse) periferal blood (namely the number of white blood cells). I collected 100µl murine blood from tail + 20µl 0,5M EDTA, then I used RBC Lysis Buffer for 10 min kept on ice (9:1- this mean 120µl blood+EDTA and 1,080 ml RBC lysis buffer). After that the sample has been washed three times and antibody for CD45+ has been added (the washing procedure followed the protocol) . Then the sample was diluted to 200µl and trasfered into the U- well, and finally 10µl of calibration particles was added (10400 of particles in one well).
When I analysed this sample I got 10 times less white blood cells than is the average number (I got only cca 600 leukocytes on 1µl and the average is 6000-7000 leukocytesµ/l). We have tried (1) to decrease a time when RBC lysis buffer interact with blood,(2) to decrease the ratio of RBC Lysis Buffer versus blood (1:1, 5:1, respectively) - but the result was still the same.
Does anyone know what could be the reason and how to solve this problem.
Thank you very much.