counting cells

[biology_06er]

Hi there,

The other day I had to count cells after resuspending my cells in 4mL. I took 10uL of cells mixed with 10uL of trypan blue and loaded onto a hemocytometer...I had soo many cells I couldn't count so I diluted my cells 15 fold (3.3uL of cells + 46.7uL of media +50uL of trypan blue)..I counted around 381 cells in the grids all up so just wondering when its time to count the total amount of cells in that whole 4mL do I use the following formula.

(381*15)*2*10^4=1.143*10^8 multiplied by 4 =4.6*10^8

seeing as i diluted 15 fold i assume i multiply the total number of cells by 15 and then because theres a 1:1 ratio of trypan blue I multiple by 2 and then 10^4 becuase one set of 16 corner squares is equivalent to the number of cells x 104 / ml and then i multipled by 4 because thats how much i resuspended in...have i got this right? I think I may have got the typan blue dilution factor wrong because when I previously counted cells I never had to dilute my cells as they were countable...so i just used 1:1 ratio..but its the same thing right?..hope im making sense!

thanks
biology_06er

[JackBean]

sorry, but 15-gfold dilution is quite stupid. Or rather, dilute 3.3 ul into any volume is quite stupid.
Anyway, your calculation is fine, although I didn't understand the 10^4 thing.

[biology_06er]

Not really...what else was I suppose to do..I diluted 2 fold there was too many cells to count..I diluted 10 fold still to many..15 fold seemed the next logical factor. I was mixing in a well before loading on to a hemocytometer so I just used useable volumes

[JackBean]

3 or 2 ul would be perfectly fine