Hello Everyone
I am using 0.25% tryspin to digest my cells for passage/seeding. However, I just notice that the cell quality is not good based on appearance (e.g. many rough cells and clusters are observed). I am troubleshooting my parameters, and I am investigating if trypsinization contributes to this kind of cell quality.
I am planning to reduce my trypsin concentration 10x (so I will use 0.025% trypsin). I talked to my colleagues, and they use lower concentrations of trypsin.
Do you mind if you can share references that support that trypsin concentration affects cell quality?
Thanks.
Hello Simon Miguel Lopez
I may not be able to share references, but I can share the protocol which I follow to trypsinize HEK293 cells.
Use 0.05% Trypsin/EDTA to trypsinize HEK293 cells. To weaken the interactions, EDTA is frequently included with Trypsin which chelates the divalent cations such as Ca2+and Mg 2+.
You may follow the procedure given below.
Gently rinse the cells with balanced salt solution without Ca+2 and Mg+2 ions and remove the solution. Then add an appropriate quantity of pre-warmed trypsin/EDTA solution to the side wall of the flask. Gently swirl the contents to cover the cell layer and incubate the flask at 37 degree C for 2-3 minutes. Observe the cells under the microscope. If less than 90% of cells are detached incubate the flask for another 2 minutes. Just tap the sides of the flask for complete detachment and add complete culture media containing 10% FBS. Take care not to expose cells to trypsin beyond 5 mins. You may centrifuge the cell suspension at 1500 rpm for 5-10 mins to obtain the cell pellet.
I may want to ask you, how old are your HEK293 cells? If they are of a higher passage number, then probably this could happen.
Best.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line