Hi, I am trying to culture MEFs to make a stock of Feder cells. These cells were purchased from Biolabs. Unfortunately, most of the cells died, and only a few were able to attach and were harvested. Recently I plated these cells at 10,000 cells/cm2 in 0.1% gelatin-coated 75 cm flasks in MEF media (DMEM Glutamax, 10%FBS, 0.1mM MEM Non-essential amino acid, 1% Pen/Strep). I see around structure on top of these cells and couldn't figure out what it is? Can anyone help me with this? Are these OK for inactivation and later on used as a feeder layer, or should I get a new one. These are P6 cells.
Somehow, I tried a new vial of CBA-310 MEFs, but the cells died and not even single-cell attached although they say that these cells don't need gelatin, but I tried both ways and used my old stock of MEFs under the same conditions, and those attached normally. What could be the reason?
Dear Asima!
Your cells look quite healthy. If you are confused by the round structures at the top of the cells, these are also cells, but not attached. These round cells in small numbers do not interfere with the growth of the rest of the attached cells. when the cells are attached, you can carefully remove the medium and replace with a new one, along with the medium, unattached cells will be removed. Apparently the gelatin pad is right for your cells.
your MEF cells look very confluent. May be they are dying because of the over-confluency?! Try maintain them around 60-70% of confluence instead of 85-95% of confluence. Cells would die out of over-confluence. next time when you passage, try 1:10 ratio split. Round cells could be either died cells or dividing cells.
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