Hi Can u help me for counting cells by use of neubars chamber. I am currently working on a protocol on culture of macrophage from whole blood. It is something like this:(i m initially deal with 20 ml blood for practice) From the 20 ml blood, buffy coat is removed.(800*g,15 min.,R.T) then PBS is added (1:2) Overlay on the histopaque (1.083)(sigma) at equal amount. centrifuge.(800*g,15 min.,R.T) MNC ring is collected. Add 3 volume of PBS and centrifuge with 250 G for 10 minutes, RT. supernant removed and pellet collected. (2 times wash). now, i resuspend pellet by adding 4 ml PBS/ MEDIA. then again 1:20 dilution is done by using WBC diluting fluied with thoma pipette(white bid) and by neubars chamber i got 107 cells (counting 4 WBC quaters) now,wht is the my cell concentration in pellet? I got result variation by using different formula. Can u help me ?(according to my calculation it is around 21*10^6, is it true?) There is an issue about contamination RBCs, so how to get rid of them? thanks.