# Trypan blue cell counting - (Mar/23/2005 )

What is the working concentration of Trypan Blue you use and do you have a good protocol for it? It is for cell counting.

Thanks.

0.4%... or just straight out of the bottle.

yes, it's for cell counting on the haemocytometer (sp?). live cells turn blue, dead cells don't .

what i do is, put 10uL trypan blue in a 1.5mL tube. add 40uL of cell suspension, wait for a few minutes (so the cells can dye)... then pipette this into the chambers of the haemocytometer, and count. and then do the mathamagics with the dilutions, and the number of squares, and averages etc.

yes, it's for cell counting on the haemocytometer (sp?). live cells turn blue, dead cells don't .

what i do is, put 10uL trypan blue in a 1.5mL tube. add 40uL of cell suspension, wait for a few minutes (so the cells can dye)... then pipette this into the chambers of the haemocytometer, and count. and then do the mathamagics with the dilutions, and the number of squares, and averages etc.

If i use 0,4% what should i dilute it with? What do you think is best; use 0.4% or straight from the bought bottle, but then would not the concentration be too much with the last one?

Thanks.

Just was wondering....one does not necessarily have to use Trypan Blue for cell counting, correct? I count cells all the time and never use Trypan blue. What would be the reason/advantages to using trypan blue versus not?

Trypan blue tells you if your cells are alive or dead. Dead ones are blue while alive ones are white.

Hi

Trypan blue counting of cells is useful when you need to know live cell number, for instance in expression studies, where the amount of protein expressed needs to be normalised against something, so that results are comparable between experiments/cell lines, and the easiest normalisation factor is live cells number.

Bob

I use a 1:50 concentration of trypan blue, then add 50ul of cells to 450ul of TpB. (1:10 dilution). Then using a hemocytometer, if you count 160 cells, you really have 16million/mL

We always dilute 0.4% trypan blue 1:1 with cells/PBS.

My usual protocol is 0.5mL trypan blue, 0.2mL cells and 0.3mL PBS. This gives a 1:5 dilution for the math... We use larger volumes to get a more accurate count. I also count a total of 16 squares ie: do both sides of the hemacytometer count then clean and count both sides again....