I am working with Caco-2 cells for the first time. It seems that they are very clumpy and do not separate well even after passing through the stripette many times. It makes it difficult for me to count cells using the automated counter. Any ideas on how to separate them well?
My advices are:
1. Be sure you wash your cells with PBS Ca/Mg free to fragilize the cadherins.
2. Use fresh trypsin/EDTA 0.25%.
3. Increase your incubation time in trypsin until you have good cell separation.
4. Keep your cell passaging less than 70% confluency otherwise you want them to form monolayers and have them undergo differentiation.
Do these cells grow adherent? If yes, do you use trypsin or how do you get them off the surface?
Cells are adherent......I generally tripsinize after 3-5 days
Inamul.....so what should I do.....should I work again with an earlier cells passage number
I use the nexelcom cellometer....u add sample and put it in counter.......it starts counting immediately
3-4 minutes removes adherent cells.........Does waiting for 15+ minutes damage the cells in anyway?? I dont know about this.....
My advices are:
1. Be sure you wash your cells with PBS Ca/Mg free to fragilize the cadherins.
2. Use fresh trypsin/EDTA 0.25%.
3. Increase your incubation time in trypsin until you have good cell separation.
4. Keep your cell passaging less than 70% confluency otherwise you want them to form monolayers and have them undergo differentiation.
All suggestions by Abraham Alahmad are good. In addition, when resuspending you might want to do it in complete medium. And I usually pass them 4-5 times through syringe (22 gauge).
I agree with Abraham advices with emphasis on the time of incubating cells in trypsin, 3-5 minutes are enough for dissolving the adhesive proteins. Add trypsin-EDTA solution and incubate at the appropriate temperature. Check the progress of cell dissociation by microscopy. To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for them to detach. If cells formed clumps after dessociation ,You can use these guidlines.
Are you culturing them in transwells for permeability assays? If so they will appear clumpy but need to be left as is. Also to let them differentiate completely you culture for 21 days. If your plating them directly onto cell culture plates then shake the plates vigorously. Also try plating directly in the center of the well. The cells will dissociate based on centrifugal force. I would avoid trypsinizing for too long since trypsin can cleave membrane proteins.
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