Problem with seeding density - (Feb/10/2011 )
hey guys! i have a question. sometimes the seeding density for my cell culture is too low. are there any ways to increase the plating efficiency? (without changing the seed density).. thanks!
You could try conditioning medium - add medium to a growing flask of cells for 24 hours, remove and filter the medium, add to your cells that you are trying to enhance growth in.
hmmm, can u explain in detail? why we have to put in the medium for 24hrs and filter the medium and add the medium back into the culture again? thanks alot! noobie here
Cells secrete stuff (growth factors, waste products etc.), while they are growing. When you put cells into fresh medium, it contains none of these things, so it takes a while to build up a level which the cells like to grow in. Some cells will not grow well without these components, especially if they are seeded at a low density.
The 24 hours is so that the cells which you are using to condition the medium don't use up all the nutrients, but still have secreted some growth factors into the medium. The filtering is so that you don't have cells from another cell line ending up in your culture causing a contamination, which is a big problem... there are estimates that approximately 1/3 of cell cultures are actually variants on HeLa from these sorts of contaminations, or to prevent cells in the conditioned medium stuffing up your seeding densities.
Conditioned medium does not keep well, store in the fridge for a few days or frozen at -20 for about a month.