Hello Aurora Vilardi

The dissociation procedure may have been harsh causing release of genomic DNA from lysed cells. Either the pipetting may be vigorous, or the dissociating solution may be too strong.

You may add a drop of sterile DNAse (1 mg/ml in water) to the cell suspension to break down the DNA strands. You need to treat the cells more gently during pipetting, shorten the incubation period, use a weaker dissociation solution (lower the enzyme concentration).

Hold the cell suspension on ice if there is going to be a delay between removing the cells from the flask growth surface and seeding into a new flask. Be sure to use gentle centrifugation (10 minutes at 125 x g). Do not centrifuge the cells too hard or too long when removing the excess dissociation solution.

Also make sure you wash your cells with PBS (Ca/Mg free). Passage your cells when they are 60-70% confluent during maintenance in culture. Do not agitate the cells by shaking the flask during the dissociation process. Keep observing the cells periodically under the microscope during cell dissociation.

Best.

Hi, the centrifugation part is missing between steps 3 and 4. This indicates that the TrypleExpress solution remains with the cells, causing clumsiness.

Have you tried to resuspend the cells in a Ca and Mg free medium?

Hi Aurora Vilardi

Previously, I found many researchers using TrypleExpress to detach Caco-2 cells and at the same time I used Trypsin-EDTA (0.25%) for the same purpose but I faced the same problem that you faced.

At the end, I found that when counting these cells, two things must be taken care of:

1- The confluency should be between (70-90)%.

2- After make dilution with complete growth media, pipette up-down the cells with the bottom of the flask until you see the clumps dissociate. (It takes from me 2 - 5 min.)

Hope this could help you

Best regards

Hello, I found that using a 5ml pipette tip breaks up the clumps easiest!

I also use .25% Trypsin, which works well for our lab for multiple cell lines.

Thanks everyone for the answers. I needed to give it a try to understand which tip worked better. I found that both 0.25% Trypsin and the TripLE Express work fine, the hard step was to pipette up and down vigorously - the 5 ml pipette is the best one - without making bubbles. I finally managed this way!