I am working with the immortalised N/TERT1 keratinocyte adherent cell line and I need to perform a wound healing assay on them in the 96well black/clear bottom plate. Previously I was culturing them in DMEM high glucose media with supplements before the start of the experiments and later on I shifted to EpiLife media which is said to have a better growth effect on this cell line. I am seeding quite a higher amount of cells/well so that I can start with my assay on the 4rth/5th day max. after culturing, once they attain a monolayer confluency. But even on the 4rth day, the cells haven't reached 50% confluency in most of the wells. I always seed them in the centre wells so as to avoid any evaporation of culture media. This issue has remained consistent for the last 5-6 tries or maybe even more and now I am not sure whether I should blame my pipetting skills or some invisible dark force! What could be the possible reasons behind this? Does passage number effects the immortalised cell line's behaviour or is it the type of plate I am using? Earlier I have used the 96-transparent well plates but as because I am using irradiation conditions for the scratch assay, I started seeding them in the black/clear bottom plates. Also, I use antibiotics and serum in my media while culturing. I haven't found any bacterial contamination yet, so I am not sure whether we can rule out this cause.
Hello Debasmita Banik
Have you first adapted the immortalised N/TERT1 keratinocytes to the new medium (EpiLife medium) ?
When cells are first introduced to a new medium (EpiLife medium) , they will typically exhibit an extended lag phase after seeding, slower maximum growth rate, and lower maximum cell density. With continued passaging in the same new medium (EpiLife medium) the lag phase will shorten, maximum growth rate will increase, and there will be an increase in the cell density. With subsequent passages, the cells will exhibit the same grow kinetics in the new medium (in fact better as mentioned by you) as they used to exhibit in the original medium (DMEM high glucose medium).
So, I would suggest let the cells get adapted to EpiLife medium for a few passages and then use these adapted cells to perform the wound healing assay.
Good Luck.
I have actually adapted the cells in the new media for 2-3 weeks ( 3-4 passages) before I seeded them for the experiment. As you said, the cell density definitely took time to increase but now they are growing well in the T-75 flasks which I use for passaging.
Hello Debasmita Banik ,
Generally the growth medium of keratinocytes (such as EpiLife) has low concentration (1.5 mM) concentration of CaCl2. Calcium is critical for the formation of monolayers as it enhances intracellular adhesion and promotes the differentiation of keratinocytes. You may try to add CaCl2 to EpiLife medium to match that in DMEM to see if it helps.
CK
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus