Cell counting (hemocytometer & automated)
Accurate cell counts are essential for reproducible transfection, viral titre, and quantitative experiments. Trypan blue distinguishes live (excluded) from dead (blue) cells.
Hemocytometer
- Trypsinise and resuspend cells thoroughly to a single-cell suspension.
- In a fresh tube: mix 10 µL cell suspension + 10 µL 0.4% trypan blue (1:2 dilution).
- Pipette up and down to mix. Load 10 µL into the hemocytometer chamber by capillary action under the cover slip.
- Under 10× microscope, count cells in 4 corner squares (each = 1 mm × 1 mm × 0.1 mm = 0.1 µL = 10⁻⁴ mL).
- Count cells touching the top and left lines; skip those touching the bottom and right (so each cell is counted once).
- Calculate:
Example: average 50 cells/square × 2 (trypan dilution) × 10⁴ = 1 × 10⁶ cells/mL.cells/mL = (mean count per square) × (dilution factor) × 10⁴ - Multiply by total volume to get total cells.
Automated counter (Countess, LUNA, etc.)
- Mix 10 µL cells + 10 µL trypan blue.
- Load 10 µL into a counting slide.
- Insert into instrument; focus; read live + dead counts plus viability %.
- The instrument already accounts for the dilution if you set "1:2".
Common seeding densities (HEK293T, 10% FBS)
| Vessel | Working vol. | For ~70% confluence in 24 h | For ~80% in 48 h |
|---|---|---|---|
| 96-well | 0.1 mL | 3 × 10⁴ | 1.5 × 10⁴ |
| 24-well | 0.5 mL | 1.5 × 10⁵ | 7 × 10⁴ |
| 12-well | 1 mL | 3 × 10⁵ | 1.5 × 10⁵ |
| 6-well | 2 mL | 6 × 10⁵ | 3 × 10⁵ |
| 10 cm dish | 10 mL | 3 × 10⁶ | 1.5 × 10⁶ |
| 15 cm dish | 20 mL | 7 × 10⁶ | 3.5 × 10⁶ |
Tips
- Pipette the cell suspension thoroughly just before loading — cells settle in seconds.
- If cells are very dense (> 5 × 10⁶/mL), pre-dilute 1:5 or 1:10 in PBS before adding trypan.
- Trypan-positive cells should be < 10% for healthy cultures.
- Clean the hemocytometer with 70% EtOH after every use, dry with a Kimwipe.