Counting B cells in hemacytometer - (Oct/12/2008 )

Hello everyone!

I am working on B-cell lines and I always count my cells before splitting them or before any treatment. I would like to ask you whether my way of doing so, is correct:

1.I take 10microliters of my suspension and add it on the hemacytometer
2.Count the four big squares
3.Take the average of these 4 big squares and
4.Multiply it by 10.000. The number I get is the number of cells/ml

So, I would like to ask you, if this procedure is correct, and why so I take 10 microliters and not another quantity??

Thank you very much in advance!!

The method is same as counting lymphocytes.

U might be taking 10ul because that is the approximate volume of the hemocytometer.

The number of B-cells will depend upon the volume of cells suspension U r taking (and the dilution of the cell suspension also if U r using Trypan blue also.)

Number of cells = average of 4 big-squares X dilution(if diluting) X volume of suspension

the suspension should fill the squares uniformly and should not flood the slide nor fall short.

It might be that U r taking 10ml of the suspension and for that reason multiplying by 10.

Number of cells = average of 4 big-squares X 10

Please see this thread and there is one PDF of cell-counting posted at 4th or 5th reply.

http://www.protocol-online.org/forums/inde...ost&id=2285

Thank you very much for yr immediate response!!!