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counting cells before adding them to 96 well plate - (Aug/23/2012 )

Hi all,

can any one please help explaining the hemocytometer and specially the calculation part, AM REALLY LOST.

am using HepG2 cell line which is cultured in petridish and I need to have 5000 cell/ well .



Assuming the hemocytometer is like the one I use....

There should be a grid on the hemocytometer that you see through the microscope, with some bigger and smaller squares. Each square represents both a unit of area and a unit of liquid volume (because the specialized coverslip you put on it has a specific weight to it, in which only a specific amount of liquid can be under it). Based on the number cells per square, or set number of squares, you can calculate the number of cells you have in your sample, and from that calculate the volume needed to give you 5000 cells.

What I would do is find the brand of hemocytometer you have, go online and find the data sheet for it. That should tell you the units of area and volume that each square on the grid represent.

Does that help/make sense?


This is about the most common sort of question we get on here:

See the attachment to my post here





Thanx a lot for your help all.

I have another question, I did count my cells and I had total # 103 cells from 4 squares.
now, i need to have 5000 cells per well (96 well plate) to do MTS assay.
I kind of lost now in the calculation part.
concentration (cells/ml)= 103/4 x 2 (dilution factor) x 10,000 = 515000
to calculate total number of cells= conc. X vol. of sample ( I did not understand which vol.) ???
after that what I should do exactly to get the number of cells I want ??



The sample volume is the total volume you have collected your cells in - the volume you took an aliquot out of for counting. As you have a higher concentration than you need, you can dilute directly from that volume... provided you have enough cells in total. To work out how many cells you need multiply the number of wells you want to seed by the number of cells per well. Don't forget to add a couple of wells for pipette volume error.


I've attached a document showing how to adjust and determine cell density in order to plate the same number of cells in multiple vessels or how to determine the cell count after adding different volumes of medium.
Attached File


i use cell countess chamber slide and the countess(invitrogen) to calculate the viable cells. but, for the fist time i used,it show low viable cells and high dead cells. then after about 2-3 minutes, i insert the countess chamber again and it show different result. the number of viable cell increase than the first one. i am not confident with the result. is that any precaution i should consider while handling the countess chamber and the countess machine?