Prepare a Cell Suspension

1. Aspirate the cell media from the the bottle.

If culture is fully adherent: Remove and discard the cell media.

If culture is HYBRID: Centrifuge media @ 1,200 rpm for 5 min.

If culture is Suspended: Centrifuge media @ 1,200 rpm for 5 min.

2. Wash the cells with PBS 1X (25 cm2: 5mL, 75 cm2: 10mL & 150 cm2: 20mL) without Ca₂+ & Mg₂+, aspirate and discard the PBS from the bottle.

3. If culture is adherent or Hybrid: Add Trypsin-EDTA (25 cm2: 0.5mL, 75 cm2: 2mL & 150 cm2: 5mL) to cover the cell layer and incubate for 2-3 minutes,

4. Add RPMI-1640 complete medium (25 cm2: 4.5mL, 75 cm2: 8mL & 150 cm2: 5mL) and disaggregate cells by making them pass several times through the pipette against the bottom of the bottle.

5. Transfer the cell suspension to an appropriate test tube.

Cell Counting with Hemocytometer

6. Non-viable cells will appear blue and viable cells will be clear.

7. When using a hemocytometer, a maximum cell count of 20 to 50 cells per 1 x 1 mm square is recommended. If cell count per square is too low it means that cell suspension solution is too diluted, and if cell count per square is too high it means cell suspension solution are too concentrated.

8. Mix 10µL of cell suspension with 10µL of Trypan Blue in a microtube. Pipette up and down to mix cell well with the dye solution.

9. Load 10µL of the cell-Trypan blue mix into each chamber of the manual hemacytometer (do not stop loading the chamber until all 10µL are loaded)..

10. Count the cells on the four (4) corner squares of the hemocytometer chamber under the inverted microscope using the 10X objective. Repeate per chamber.. Take the average count per chamber

11. Compute the following metrics for each culture:

a) % Viable Cells

b) Cell Concentration

c) Total Cells