Hello, I need to obtain macrophages from exudate peritoneal cells for primary culture, I injected 5 ml of thioglycolate 3% in the peritoneal cavity for three days, first, I sacrificed the rats, next, I washed the peritoneum with 10 ml of  cold PBS with FCS 10%, then, I obtain a volume between eight and ten militers, after that I had to use ACK lysis buffer to lyse red blood cells, then centrifuge 1800 RPM for eight minutes, protocols report that, therefore I should  obtain 5x106 cells per militer, but my results aren´t the ones reported (1x106 cells sometimes zero¡¡), what should I do?

I will very greatful.