Hi
I just received a vial of Caco2 from ATCC. I am going to make stocks of it for next time uses. Once i revived the cells from the ATCC i found out that the cells have difficulty to attach to the substrate. I kept splitting the cells into two flasks every 24 hours. The cells from the original (first flask, day 1) started to attach after 8 days. They are forming many cell patches and not forming a nicely monolayer (Please refer to attached picture). I subs the cells after it reaches 90% confluence (1:2 ratio). Is it normal for the newly purchased cells to take some time to attach?
After subsculture, i was shocked that the cell start to confluence after 3 days. And i subs it into (1:4 ratio). I think the cells are now adapting to the culture condition?
now i have 16 flasks and decided to cryo 12 flasks of them. What is the best way to cryo it. Is it okay to add 5% DMSO directly into the cell suspension? Because as far as i know DMSO is toxic to the cell. Direct contact of DMSO to the cell may cause shock?
Or is it the best way to prepare the freezing media first. Then resuspend the cells in the freezing media?
what is the best cryo cell density (number) for caco2?
Complete growth media used : EMEM + 20% FBS + 1% pen/strep
Freezing media : 5% DMSO in complete growth media
Thank you all
Hi,Nur Shafini Che Ahmad
1.when Caco-2 cells are subcultured,it takes about 48h to attach substrate.It may takes more time for thawing cells.
2.the seed density is important for Caco-2 cells to form a nice monolayer.30000 cells per cm2 is recommended.Counting cell is necessary for every subculture.
3. it is the best way to prepare the freezing media first,then use freezing media to suspend cells.
Freezing media:5% DMSO+10%FBS DMEM
here is a protocol for culturing Caco-2 cells
Please do not add the DMSO directly to the cells! Aseptically prepare the cell freezing medium at 10% v/v of DMSO in full culture medium just prior to use. Once cells have been gently detached from their flasks, centrifuge to pellets before resuspending in cell freezing medium - no more than 500,000 cells per vial (1ml). Place cryovial immediately in a Mr. Frosty cryovial store before placing at -80C for a minimum of 12h (overnight will do). Then transfer vials to vapour phase of liquid nitrogen for long-term storage. Freezing cell should be controlled and slow (apporx. 1 degree cooling per minute at -80C) and thawed quickly to room temperature before resuspending in fresh medium once thawed. Resuspend carefully and gently to ensure single-cell suspension. It may be that your aggregates are clumps of cells from incomplete resuspension. DMSO is only toxic at RT, not when frozen.
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