Before you start
- Passage at 70–90% confluence; do not let cells overgrow.
Reagents and materials
Totals are for 2 flasks (+10% extra). Per-T75 flask amounts are in the middle column.
| Reagent | Per T75 flask | Total |
|---|---|---|
| PBS, Ca²⁺/Mg²⁺-free (wash) | 5 mL | 5 mL |
| Trypsin–EDTA (0.05–0.25%), pre-warmed | 2 mL | 2 mL |
| Complete medium (neutralise + reseed) | 18 mL | 18 mL |
Buffers and recipes
PBS (1×) per 1 L
| NaCl (137 mM) | 8.0 g |
| KCl (2.7 mM) | 0.2 g |
| Na₂HPO₄ (10 mM) | 1.44 g |
| KH₂PO₄ (1.8 mM) | 0.24 g |
| Ultrapure water | to 1 L |
Adjust to pH 7.4 and autoclave. This PBS is Ca²⁺/Mg²⁺-free (use it for detaching cells).
Equipment
- Biosafety cabinet
- Inverted microscope
- Centrifuge
- Hemocytometer or counter
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Remove medium
Aspirate the medium.
Wash
Rinse with 5 mL Ca²⁺/Mg²⁺-free PBS to remove serum (which inhibits trypsin).
Trypsinise
Add 2 mL trypsin–EDTA, tilt to cover and incubate at 37 °C for 2–5 min, until cells round up and detach with a gentle tap.
Neutralise
Add 2–3 volumes of complete medium and pipette gently to break clumps.
Count (optional)
Count cells if you need a defined seeding density.
Re-seed
Transfer the appropriate volume (for example a 1:3–1:10 split) into a new flask with fresh medium.
Record
Label with the new passage number and date.
Critical points
- Over-trypsinisation damages surface proteins and viability.
- Keep a passage-number limit for each line and use low-passage stocks.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Cells will not detach | Make sure serum was washed off; warm trypsin; use a higher trypsin concentration briefly. |
| Clumps | Pipette gently; add EDTA; filter if needed. |
Safety
Follow the biosafety level of your cell line.
Further reading
- ATCC Animal Cell Culture Guide.
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.