0.25% trypsin-EDTA recipe
The enzyme solution for detaching adherent cells: 0.25% trypsin, 1 mM EDTA. Dilute to 0.05% for sensitive cells.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| Trypsin (1:250 powder) | 250 mg | 0.25% |
| 0.5 M EDTA (pH 8.0) | 200 µL | 1 mM |
| 1X PBS (Ca²⁺/Mg²⁺-free) | to final volume |
Steps
- Dissolve trypsin slowly in Ca²⁺/Mg²⁺-free PBS (or HBSS).
- Add EDTA and check pH 7.2–7.6.
- Filter-sterilise through 0.22 µm.
- Store aliquots at −20 °C; keep thawed aliquots at 4 °C for 1–2 weeks.
Good to know
- Wash away serum first — serum α1-antitrypsin inhibits trypsin.
- Stop the reaction with serum-containing medium after detachment.
⚠ Safety Handle solutions for live cells aseptically in a biosafety cabinet.
FAQ
How long at 37 °C?
Usually 2–5 min. Beyond 10 min surface proteins suffer — watch for cells rounding up under the microscope.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.