Initially there was a flask at P13 which was growing very fast using all ATCC recommended conditions and products (EMEM + 20% FBS).
I then switched to using EMEM from Quality Biological, and used a Trypsin-EDTA that was a higher concentration than recommended so I used it for a smaller period of time during passage (3 minutes instead of 10). Once I passaged it into 3 new flasks at 1:6, it took those P14 flasks 10 days to get confluent (normally they are 90% confluent within 4 days) and cells were not equally distributed throughout the flasks.
I figured growth was hindered due to different lab conditions and passaged again thinking it would adapt. Sadly, the P15 flasks showed even worse growth. You can see in image 1 how the cells appear and where on the flask they are growing.
I am also testing the cells on DMEM because I hope to coculture the cells with HT29 cells in the future. As you can see in image 2, those cells are also only growing where the black line is drawn on the flask, and the rest of the plate appears empty.
Image 1: Caco-2 cells in EMEM + 20% FBS P15
Image 2: Caco-2 cells in DMEM + 10% FBS P15
Merry
The high concentration of tripsin is very bad for the cells, Even in short time, because produce a damage un the cell membrane.
Luis
Merry,
you also reduced FBS by half, that is usually done when you try to slow down the cell cycle. Try returning back to 20% FBS.
Ljerka
We use also Caco-2 in our lab but we use only 10% FBS in DMEM with glutamine and glucose. We split our cells 1:6 every 3-4 days and use 1xTrypsin/EDTA (Sigma) for cell passage (10 min; 37°C). Stop the trypsin reaction with medium which contains FBS.
Contain the medium the same additives? Glutamine, glucose ...
You can check your inkubator for CO2 concentration. Try to measure the concentration in the inkubator with a hand-held measuring device. Our cells didn`t grew due to a broken CO2-sensor... The incubator showed 5% but the concentration was another...
- Badges
- Science topic