Cell line: MDA-MB-231
Media: DMEM +10% FBS
I am having trouble generating single cell colonies following FACs sorting into 96 well plates. I sort one cell into each well and allow them to grow into colonies before passing them and screening them. However, I don't really know the best way to culture them in order to give them the best possibility of growing into a healthy colony. Some cells seem to grow to 5-6 cells and then die. Others grow to larger colonies and die before becoming a mature colony that I can passage.
Does someone have a protocol for growing single-cells into colonies? How long after the initial sorting into the wells should I wait to change the media? What is the best method for generating pre-conditioned media for this application (SF vs +FBS, Confluency of cells for generation of media, time of incubation before media is pre-conditioned)?
The cells attach within 24hrs, or may take a bit longer if too much time spent on FACS sorting. So give 48hrs, and change media every 48hrs. The colonies die because lack of nutrients. I usually do limiting dilution into 96 well plate and mark single cell wells immediately. Its laborious though.
The fact that you have some cells making to multiple cell stage suggests that they are fine initially and they die out later. It appears to be a cell line specific issue. Some cell lines do not like to grow without enough of their siblings around. Try to look up the literature on culturing this particular cell line and see if there are any reports on culturing conditions. Alternatively, try picking single cell colonies directly from a plate and propogate. Plate cells sparsely in a 10 or 15cm dish and let individual colonies form on the plate. Use the age old method of picking colonies using a cloning cylinder and propogate single clones. If the purpose of FAC sorting is to isolate +ve clones, you might want to do that later in a 96 well format using a fluorescence plate reader.
Hope this is helpful. Best.
I also had a similar problem with some cell lines that don't like to grow at low cell densities/clonally. One option would be to try and put them on a layer of feeder cells that would sit there but not divide, just like people do with ES cells.
We recently used this method to isolate single cell while maintaining its siblings aside. This method can also applied on non-adherent cell. If you want to get single-cell colonies, try use larger size of well. In this article we use 40um which might be too small for your purpose. Thanks.
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