growth curve for attached cells - (Feb/28/2011 )

Hello!
I would like to make a growth curve for attached cells. I trypsinize them for 3 min (200 mu liter per well) and then suspend in medium (3 ml)and count, but I dont understand how can I make sure that I took "all" the cells from the well when I count(I grow in 6 wells)? Plus how many cells should be in each square of the 9? I read between 20-50 and in other places 100-150?
Thank you so much!

You can't be sure that you took all the cells from the wells. However, if you treated each well in the same manner, then presumably you left a similar number behind each time... it is about minimising error, not eliminating it. Besides counting on a haemocytometer has an inherent error of about 15%.

See attached protocol in this post for how to count on a haemocytometer. You should count all 9 (or 16) squares and be counting about 150-300 cells per 9 each time. You should also count both sides to ensure that you have an even distribution of the cells. Cells settle really fast in suspension, so fast that if you use the same pipette load to fill both sides (e.g. draw up 50 ul and use it to fill both sides) of a haemocytometer, you will have consistent differences between each side.