I received a vial of cryopreserved LnCaP cells from a colleague and they didn´t attach to the T25 flask. After that i asked for another vial and this time i coated the flask with poly L Lys and the problem was solved. After expanding (~80% confluence) and cryopreserving the cells (RPMI FBS10% DMSO10%) i thaw these cells but they are growing very slowly and instead of being hard to attach to the flask, now they won´t detach from the poly L Lys coated flask. I was using 1ml of Trypsin 0.05%, incubating for 5-10 min at 37°C and just some of the cells detech. After seeing this y tried using TryplE from Gibco and obtained the same result.
Should i use higher concentrations of Trypsin? I am seeing this issue also with the breast cancer cell line SKBR3. Some persons have suggested me to detach the cells manually using a cell scrapper. What would you recommend?
Hi
We have some hard-earned experience with matured DC (very adhesive). Try an ice-cold DPBS (no cations) wash to cool cells and retract integrins. Then add the warm trypsin, return to incubator for 8-10 minutes on a battery-powered shaker if you have one to give mechanical shear forces. Then harvest and add media with 10% serum to neutralize Trypsin. I advise the 1x TrypLE, it's plant based and gentle
https://www.thermofisher.com/order/catalog/product/12605010
Good Luck!
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