why centrifuge before doing a cell count? - (Sep/30/2006 )

hi, i need to do a cell count. in many of the protocols i have seen i will just use trypan blue and a hemacytometer to count the viable cells. however, i have been told i need to centrifuge the cells before this and add some medium at some point. i dont understand why i need to do this or what volume of cells needs to be centrifuged. and i dont know how much medium or when this should be added?
would be greatful for any advice...

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Usely we centrifuge cells before counting to eliminate dead cells and remains (that are lighter than living cells). After centrifugation, we discard the supernatant (to discard dead cells) and add a determined volume of fresh medium (5 to 10ml). Then we resuspend the pellet and we count the cells.
The volume of added medium is not really important. But you need to know it to determine the total number of cells in your pellet (when you count, you determine the cellular concentration, so you need to know the volume to calculate the total cells number). We don't add more than 10 ml, because it's easier to resuspend pellet in a small volume.

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