I am growing mesenchymal stem cells isolated from Wistar rats bone marrow. Initially the cells were getting detached or trypsinized in the culture flasks easily. But now I am unable to detach the cells from the flask. I have tried increasing the volume and time of incubation after trypsin treatment. Even after increasing the time and volume not even a single cell is getting detached. Please suggest me something to cope up with problem or suggest any other agent stronger than 0.25% trypsin+EDTA solution.
are you using the same culture plates, no catalog number changed. That could be. Sometime all look similar but with different surface properties.
other step, give more washing of cell layer with ca mg-free PBS before adding trypsin mixutre
I used this method in my paper (enclosed:
Once 70–80% were confluent, the dishes were trypsinized with TrypLE TM Select CTS TM (A12859, Gibco® by Life Technologies) and placed in culture for next passage.
Good luck
Subhash C. Juneja Thank you so much Sir for your response. I have tried different flasks too and generally for washing I prefer Ca Mg free PBS only. only thing I haven't tried is TrypLE. So i will go with this and update if it works or not. Thank you
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