Counting cells

Submitted by kdorfman on Thu, 09/12/2019 - 16:58

Just before plating, after cold medium has been added to the trypsinized cells,

  • mix 0.1 mL cell suspension with 0.1 mL 0.4% trypan blue. Live cells will not take up trypan blue. (Use a different dilution factor if cells are uncountable)

  • inject 10 uL to one side of the hemocytometer (see image attached below).

  • Inspect at 10x.

  • Count the number of live (clear) cells in all 9 squares of the hemocyometer. (Volume = 3mm * 3 mm * 0.1 mm = 0.9 uL) (Count in 4 1mm x 1mm squares if cells are very numerous)

  • Calculate the live cell concentration (# live cells * 1.11)/1uL for 9 squares,
    (# live cells * 2.5/uL for 4 squares)

  • correct for dilution factor

If you mixeduL cell suspension
withuL Trypan blue
then your dilution factor isX.
If you count
live (clear) cells
in 1 mm x 1 mm squares
there are live cells per uL
to get live cells
you need uL