Klapperich Lab:Cell Counting Procedure

Overview
How to count cells using Hemocytometer.

Materials

 * PBS
 * trypan blue soln.

Procedure

 * 1) Aspirate media.
 * 2) Wash with 1X PBS.
 * 3) Aspirate PBS.
 * 4) Resuspend in media – make sure cells are completely resuspended, that they are not clumped together.
 * 5) Place coverslip on middle of hemocytometer.
 * 6) remove 500 ul of cells to microfuge tube.
 * 7) Add 50 ul of cell solution to 50 ul of trypan blue solution in a second microfuge tube.
 * 8) Add 20 ul of the final solution to each side of the hemocytometer. by letting a drop of liquid get taken into the slid by capillary action.
 * 9) Place hemocytometer on scope at 10X magification
 * 10) Locate center 5 x 5 grid.
 * 11) Move to higher power.
 * 12) Count all of the viable cells in the 5 x 5 grid. It is easier to count at higher power. Dead cells will be dark blue.
 * 13) Count the cells in two other 1 mm2 areas (one of the squares surrounding the center square).
 * 14) Average the values for the final count.

Contact

 * Who has experience with this protocol?