Experimental Materials

0.4 % w/v trypan blue (GibcoBRL 15250-061)

Erythosin bluish stain

Take 0.1 gram Erythrosin bluish (Sigma E-9259) and 0.05 gram preservative methyl paraben (Sigma H-3647) dissolved in 100 ml Ca /Mg free saline

Hemocytometer and coverslip

Counter

Low magnification inverted microscope

Particle Counter (Coulter counter, Coulter Electronics)

Experimental procedure

1 Mix 50 ml of cell suspension with 50 ml trypan blue (or Erythrosin bluish) in equal volume in a 1.5 ml microcentrifuge tube.

2 Take a small amount of liquid (about 15ml) from the groove above the blood cell counting tray, cover with a cover slip, observe under a 100-fold inverted microscope, live cells are not stained, dead cells are blue (or red - Erythrosin bluish ).

3 Count the total number of cells in the four large squares, divide by 4, multiply by the dilution factor (at least multiplied by 2, mixed with the volume of trypan blue), and finally multiply by 104, which is the number of cells per cell suspension. . If the cell is on the line, count only the cells on the line and the right line (or the cells on the left and left lines).

4 large cells * total number of cells x 2 x 104 / 4 = number of cells / ml * volume of each large cell = 0.1cm x 0.1cm x 0.01cm = 10-4 ml

4 If you do not use the blood cell counter, you can use the Coulter counter to automatically count, but you can't distinguish dead cells or living cells.

5 Example: T75 monolayer culture Make 10ml cell suspension, mix 0.1 ml solution and 0.1ml trypan blue in a test tube, add a little mixture to the hemocytometer, and count the number of cells in the four squares.

Number of living cells/square: 55, 62, 49, 59
Number of dead cells / squares: 5, 3, 4, 6
Total number of cells = 243
Average number of cells / square = 60.75
Dilution factor = 2
Cell number/ml: 60.75 x 104 x 2 = 1.22 x 106
Cell number /flask: 1.22 x 106 x 10ml = 12.2 x 106
Survival rate: 225/243=92.6 %

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