Page 8 - Cell Culture Training Hand Book - A4 Edited 20.1.2020

CELL COUNT & CELL VIABILITY

Reagents
+2
+2
4. DPBS 1x (Dulbecco’s Phosphate Buffered Saline) (- Ca - Mg )
5. Trypsin EDTA 0.25%
6. Culture media (DMEM with 10% FBS)
7. Trypan Blue

Protocol
1. Discard media from the flask.
2. Wash cells with 5 ml of DPBS (- Ca - Mg ) for 2 times.
+2
+2
3. Add 2 ml of trypsin to detach the cells.
4. Incubate the culture for 5 minutes at 37°C.
5. Tap the flask gently, then observe the culture under microscope to ensure cell detachment.
6. Add 4 ml of culture media to neutralise trypsin.
7. Transfer the mixture into fresh 15 ml falcon tube.
8. Observe the flask under microscope to check if cells have been removed completely.
9. Harvest the collected mixture by centrifugation at 1,500 rpm for 5 minutes.
10. Remove the supernatant. Tap tube gently to loosen pellet and resuspend the pellet (at least
50 x, and avoid bubbles) with 1 ml of culture media. Volume of culture media is in
accordance with cell pellet.

11. Take 10 µl of the cell suspension and mix well with 10 µl of Trypan Blue (ratio 1:1) to
stain the cell on a parafilm.

12. Prepare hemacytometer . Load 10 µl of stained cell under the coverslip of
hemacytometer. Suspension will be drawn under the coverslip by capilary action.
13. View slide with 4x magnification.
14. Use hand-held counter to count cells in each of the four corner (1, 2, 4 and 5 in diagram
below) in the counting chamber.

Tips
1. Make sure cells are well suspended to avoid clumps during cell
count.
2. Clean the apparatus and cover slip with 70% ethanol. Careful
handling of the apparatus is necessary to prevent damage to the
glassware and injury to the worker.

15. Calculate cell number by the following calculations:
Formula
4
Cells/ml = average count per square × dilution factor × 10
4
Total cells = average count per square  dilution factor  10  volume of cell suspension

CELL VIABILITY

1. Count total number of cells and total number of viable (unstained) cells.
2. Calculate percent of viable cells as follows:
number of unstained cells
% viable cells =  100
total number of cells

Cell Culture Training Hand Book | CMBL Page 8 of 12