Hello, I have been using 1x trypsin (0.25%)/EDTA to detach the cells. However, it took me over one hour and lots of flask rocking/banging but still most of the cells were still attached to the flask. Also I noticed cell clumping. I tried to use 10x trypsin without EDTA. It did work after about 10 minutes with high percentage of live cells. However, 10x is very strong and cells might be stressed. Anyone has similar problem? How could I improve this? Many thanks.
Hello Hongjuan Zhao
You may try doing the following.
1. Wash the cells with PBS without Ca2+/Mg2+ (because calcium and magnesium facilitate cell binding and clumping) about 2-3 times and again give a brief rinse with warm trypsin/EDTA solution.
2. Do not increase the concentration of trypsin as it may be detrimental to the cells. However, you may increase the volume of trypsin if required.
3. Carry out the trypsinization process by incubating the cells at 37-degree C in the CO2 incubator for maximum 4-5 minutes.
4. The most important factor is trypsinization of cells when they are 70-80% confluent. When cells are more than 80% confluent, it becomes difficult to trypsinize the cells, especially some of the keratinocytes. So, make sure that your cells do not reach confluency when you are about to trypsinize them.
5. As far as possible work with cells of lower passage.
Best.
Thank you very much. I have tried all of your suggestions but only 10x trypsin worked. That's why I am using the higher conc. of trypsin but worried about the stress to the cells.
Maybe our HaCaT cells have changed. The stock culture was from one of our PhD student, who also used 10x trypsin.
Malcolm Nobre
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