I was trying to freeze some cells. my cells were like 80% confluent in the flask and I trypsinized them, tried as much as I can to avoid clumpes by drawing cells up and down several times and then took 10 microml of the solution on each side of hemocytometer. However I got very few cells when I counted ( average was 50x10^4). what went wrong in the process for me to get this few number of cells ?
I am using UM UC6 bladder cancer cells.
When you re suspend the cells after trypsanisation and before counting make sure that you reduce the volume of media added to the cells. That way your cell suspension will have a higher cell number.
Alternatively spin down your cell suspension and just resuspend in a smaller volume.
Finally you could try and grow two flasks of cells and combine them together before you do your cell count. That way you will have a greater number of cells to start with?
Normally what I do is centrifuge the cells after trypsinization. Then resuspend the cell pellet in 1 mL of medium and take 15 uL of suspension to mix with 15 uL of trypan blue. The cell number is high for 2 X dilution. Ensure the cell suspension is homegenous when you pipette out the volume. Otherwise may cause fewer cell count due to technical errors.
Did you look at the flask you were growing them in. Sometimes all of them do not detach.
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