I have performed a 10 day long treatment with MCF-7 cells in 24-well plates. I have senesced them, and followed by treatments with drugs that are expected to kill normal cells, but also have senolytic properties. I have seen contamination on other plates, but the morphology of those were extremely noticeable, the media was yellow, and the MTS assay numbers were through the roof (I presume because of the contaminants). These cells however have a normal media color (DMEM + 10% FBS), mostly normal morphology, and the MTS absorbance values were significantly less than the untreated cells. They just have loosely attached aggregates of darkened clumps of cells that look almost spheroid-like (see photos; first three are 4x, last three are 10x). They aren't freely moving, but only wiggle a bit without breaking apart whenever you move the plateI have to take these to be visualized for a different procedure, but don't want to take them contaminated wells. Am I being paranoid?
Upon browsing, I found that these structures look extremely similar to MCF-7 mammospheres.
Hello Joseph
Did you refresh the medium during the entire period of 10 days? To me, it looks like nutrition depletion.
Hi Joseph,
it looks like that your cells were forced to grow up to extremely high density and have detached. Lack of attachment drives the cells to aggregate and form spheroids. The yellow color of your medium also indicates rather high acidity, which may lead to cell detachment / death. This could also explain the increase MTS absorbance. I'm not sure I understood everything of your protocol, but if you want to keep your MCF-7 cultures in a stable state for performing subsequent experiments, I recommend reducing the cell numbers, avoiding long term conflurence /high density as well as medium depletion.
Yes. I treat the cells at about 60% confluence in a 24-well plate. I then changed to fresh media after 24 hours when removing the first drug. On day 7 I treated with second round of drugs in fresh media, which were presumed to be cytotoxic to normal cells and had unknown effects on senesced cells. At this time point there were large populations of senescent cells, but the normal growing cells were extremely confluent. I changed the media again after 48 hours of exposure to the second round of drugs, which is when I began to observe this.
The yellow color was only in wells with what appeared to be floating sheets of cells (but you could not clearly make out the cell type). The media change came just 24 hours after adding fresh media, and the MTS readings for these wells were WELL above a normal confluent untreated well. In my experience this seemed to me like contamination, and I threw these samples out. These on the other hand do not have the floating sheets of cells, only the spheroid looking clumps and other dead floaters, along with some senescent cells and some normal attached cells. The media is normal and the MTS readings are significantly lower, so I assumed there was no contamination.
Hi again.
if you can exclude bacterial contamination (which you could see at higher magnifications - moreover, the medium would become turbid), check for mycoplasma.
Angela
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