I am a new person in animal experiments and usually, for cell counting, I use an automated cell counter. but, for this experiment, because the mice were infected I want to do counting manually.
after the separation spleen of mice and using the dissociation kit and making the cell suspension, I want to count the number of total cells. usually, I take 25 µl Cell suspension and 25 µl trypan blue for counting the cells with a hemocytometer.
cells/ml= the number of cells/4 * 2* 104
Then=> if I want to know the number of cells in total volume => cells/ml * total volume
in animal experiment, there are a lot of cells that is really hard to count them.
what should I do?
I think, first I can take 100 µl spleen cell suspension and 900 µl PBS=> then count the cells in this 1 ml. through this formula:
cells/ml= average number of cells * 2 * 104
then for getting the number of cells in original cell suspension ==> cells/ml * 10 (pre-dilution factor)= gives me the number of cells in 1 ml of original spleen cell suspension.
then if I have 5 ml total volume I can multiply 5 to get the total number of cells in 5 ml from a spleen of mice.
Am I right or something is wrong?
Hello Poorya Davoodi
Calculation is right, but the way of doing it is incorrect.
Pipette 25ul of cell suspension from 5ml spleen cell suspension and dilute it to 1:10 (Say Solution A)
Take 25 µl of cell suspension (Solution A) and 25 µl trypan blue for counting the cells with a hemocytometer.
Use the formula
Cells/ml = (Total cells counted x Dilution factor (2) x 10,000 cells/ml) / Number of squares counted --- (Say X)
Cells/ml in the original cell suspension = X x 10 (pre-dilution factor)--(Say Y)
Total cells in the sample (5ml) = Y x total volume (5)
Do not take 100 µl spleen cell suspension and 900 µl PBS=> then count the cells in this 1 ml. Instead do 1:10 dilution of the 25µl cell suspension taken from the original 5ml spleen cell suspension.
Best.
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