My cells did not detach during subculture. What could be the reason?
If cells do not detach or detach very slowly during subculture, this is usually caused by suboptimal subcultivation conditions or handling.
Possible reasons and solutions
Please note: Some epithelial cell types adhere very firmly and need prolonged incubation times. To avoid irreversible damage by over-trypsinization, the use of accutase is recommended. Accutase is very gentle to the cells, preserves the surface receptors and doesn’t affect the cellular viability during extended contact with the cells.
Possible reasons and solutions
- Cells are too confluent → Trypsinize cells at a subconfluent stage
- Washing step was omitted → Perform a washing step with HepesBSS or PBS to remove residual medium before adding trypsin
- Trypsin solution is too cold → Use pre‑warmed trypsin (room temperature or 37°C)
- Trypsin solution is too old → Check the expiry date and use a fresh solution
- Incubation time too short → Use proper incubation time until 80% have detached
Please note: Some epithelial cell types adhere very firmly and need prolonged incubation times. To avoid irreversible damage by over-trypsinization, the use of accutase is recommended. Accutase is very gentle to the cells, preserves the surface receptors and doesn’t affect the cellular viability during extended contact with the cells.
