Counting cells - To spin or not to spin... (Apr/09/2008 )
Hi, i have a problem with the counting of cells in tissue culture.
Its not with the hemocytometer but with the protocol itself.
The protocol i was given say that i need to spin my cells upon trypsin neutralisation. And so i did. I then resuspend the pellet with 5 ml of medium. However, upon mounting it on the hemocytometer, i saw that the cells were too many to count. I diluted again to 10mls and still it was too many. I ask the person who gave me the protocol and she said, she only estimated the cell count, no cell counting.
Is it possible that by spinning the cells and the number of the cells i got is high, would that be false and not a representative of the true number? If so, can i not spin it? I am thinking of not spinning it later to see how it goes.
Its not with the hemocytometer but with the protocol itself.
The protocol i was given say that i need to spin my cells upon trypsin neutralisation. And so i did. I then resuspend the pellet with 5 ml of medium. However, upon mounting it on the hemocytometer, i saw that the cells were too many to count. I diluted again to 10mls and still it was too many. I ask the person who gave me the protocol and she said, she only estimated the cell count, no cell counting.
Is it possible that by spinning the cells and the number of the cells i got is high, would that be false and not a representative of the true number? If so, can i not spin it? I am thinking of not spinning it later to see how it goes.
Dear Tkhaw,
In practice there should be < 100 cells/square (16 small squares) to make counting easy.
This method is qualitative i.e. you can get 10 people to count the same cell suspension and get 10 different cell numbers. HOWEVER THEY ALL SHOULD BE WITHIN < 5% of each other.
If they are not then you have a problem.....either with cells being counted that should not be counted (as they are on an edge of a square) or having a non homogenous cell suspension.
If the cell suspension cannot be diluted then you have 2 choices:
i) Only count 2 small squares/ large square (4 in total)
ii) Take a very small sample of cell suspension and highly dilute
Either option can INTRODUCE GREATER ERROR.
If you take a sample of cells prior to centrifugation this can also cause problems, especially if you are doing VIABLE counts. The viable count pre and post centrifugation will be DIFFERENT......centrifugation, even at 100g, will cause cell death.
If you can afford either the cost and the loss of cells, BUY a PARTICLE COUNTER....what used to be called a coulter counter. These give you accurate and precise cell numbers, that can be compared between experiments. We have a Z2 particle counter from Beckman Coulter.....well recommended.
Hope you find this useful
Kindest regards
Rhombus