Hii, Could anyone please tell me if my calculation sounds right or wrong
Lets say, I have counted 500 cells in the 4 side squares of neubauer chamber and have done 1: 10 dilution with trypan blue
then, Cells / ml comes out to be =#cells/ml= [total number of cells counted (500) divided for the number of square counted (4)] multiply for dilution factor (10) multiply for chamber factor 10^4
500/4= 125 ; 125x10x10^4=12500000 cells/ml
My original volume in which cells are suspended is 500 ul, then
Total number of cells in 500 ul = 6250000 cells
And, if I want to plate 60,000 cells per well, then I should put 4.8 ul of the original cell suspension per well .
I'm calculating in another way, but maybe this helps you: I'm countin the four big squares and make the median, then I calculate:
median: 125 cells per square --> 125 cells/0,1µl --> 1250 cells/µl in your suspension
if you want to plate 60.000 cells, that means 60.000/1250 = 48, so you need 48µl of your cell suspension and you don't have to take care about factors and volumes ;-)
oh sorry, I forgot the Trypan Blue dilution - so you have to take 4,8µl of your suspension!!
The calculations are fine, but I would be concerned about a 1:10 dilution in Trypan blue. Often Trypan Blue is sold at 0.4% The final concentration for manual counting should be at 0.1%. Generally I recommend that people make a 0.2% stock by diluting 1:2 in pbs and filtering to remove the debris that is often in the factory bottle, then they can do a 1:1 mixture with their cell sample. Why is this important? Trypan blue is cytotoxic and too high of a concentration can lead to erroneous viability measurements.
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