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Difficulty in doing hemocytometer cell count


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#1 SStfter

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Posted 13 May 2009 - 05:51 PM

Hey guys,

I'm having difficulty counting my cells with a hemocytometer. I can do this fine for L-cells, SF9 etc but these 293E suspension growing cells just don't want to be counted. I make sure they are resuspended properly at each step (they like to clump but dissociate easily enough) but when it gets to loading them onto the hemocytometer, they don't seem to correspond to my original sample. Eg. I split them 3 days ago to 1.5x10^5 and when I counted them today they were at 4x10^5. Looking at the cells in the flask through the microscope they are easily 1x10^6 (ie. what they should be).

Anyone have any tips? Are the cells sticking to anything?

Thanks,
Seb

#2 bob1

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Posted 14 May 2009 - 04:26 PM

how much of the stuff in the flask is dead? Did you resuspend them properly?

#3 SStfter

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Posted 18 May 2009 - 03:51 PM

how much of the stuff in the flask is dead? Did you resuspend them properly?


I resuspend them properly. I pipette them up and down with a sterile glass pipette (gently) and look at them under the microscope before taking an aliquot for counting. I tend to have around 95% viability, sometimes better sometimes worse.

#4 bob1

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Posted 18 May 2009 - 05:17 PM

cells settle really fast, as in I have seen consistent changes in cell number between the different sides of the haemocytometer when both sides are loaded with the same aliquot of cells (as in sucked up enough to load both sides and used that), with a time difference of less than 20 seconds. Could it be that you are letting your cells settle before loading the haemocytometer?

#5 Stephan

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Posted 01 June 2009 - 03:02 AM

cells settle really fast, as in I have seen consistent changes in cell number between the different sides of the haemocytometer when both sides are loaded with the same aliquot of cells (as in sucked up enough to load both sides and used that), with a time difference of less than 20 seconds. Could it be that you are letting your cells settle before loading the haemocytometer?



I agree with bob, I was counting cells the other day and they adhered within 10min so they may become "sticky" within seconds. If you are using trypan blue for a viable cell count - make sure you pipette up and down again before taking out the sample and just work as fast as possible.

good luck.




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