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In the last days we have been discussing a very trigger question in our lab about cells counting. We realize that most of our co-workers didn't know how to count cells.
What is your opinion?
We have a cell culture with 20mL.
We pick up 1mL to and eppendorf and ressuspend.
From that we pick up 10uL to a new epp and then we add 90ul of tripan blue.
From this last mixture, we pick up 10uL to a hemacytometer counting chamber.
We count something an average of 58 cells in 1/16 squares.
Question: what is the final concentration p/ mL?
Re: Cell Count
I do not know the hemocytometer used in your lab. I use the one which has 25 small squares in center. And I can tell you the way for us to get the cell concentration:
(1) Get 10Ál cell resupension to a hemocytometer counting chamber like you did. 10 Ál is required by the hemocytometer.
(2) Count cells within the 25 small squares (for example, 58 like what you got).
(3) The surface area for each small square is 1/25 mm squared, and the depth of chamber is 0.1 mm. So that the volume for all 25 small squares is 0.1 mm cubed.
(4) Now, we get the cell concentration:58/0.1 mm cubed, or 580/ 1 mm cubed.
(5) And we know 1 cm cubed ( 1 ml) equals 1000 mm cubed.
(6) So the cell concentration is 58*10*1000/ ml
Please I think you can get the way to calculate cell concentration from your haemocytometer's manual.
|The Following User Says Thank You to wangleipurdue For This Useful Post:|
Re: Cell Count
Thanks for your response!
A question: what is the origin of your *10 of your formula.
In my calculations, I used a dilution factor in Triptan Blue: 10ul of sample to 90 of solution. So in my formula I have = 58*10*1000/mL.
But in your description, I can see a dilution factor...or I could be wrong in my formula (also)
Re: Cell Count
In my formula, there is no dilution factor, please refer to step (3) and (4). Because the volume for my hemocytometer is 0.1 mm cubed, so that what I get 58 from counting, that means 58/0.1 mm cubed, in other words, 58*10/ 1 mm cubed. And because 1 mm cubed is 1 Ál, and 1 ml equals 1000 mm cubed, so that the final concentration is 58*10*1000/ml.
The way I showed you is to get the concentration of any solutions that you put into the hemocytometer, if you dilute your solution before adding samples in hetocytometer, please multiply it by dilution factor after you get the concentration of any solutions that you add direct into hetocytometer.
Once again, my formula has no dilution factor, it is to get concentration of any solutions that you add into the hemocytometer.
|cell , count|
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