I am afraid that 1 cell alone would survive very difficult. Any recommendations?Should I use a medium rich in growth factors? To use a mix of old and new mediums? eg 50% of old medium and 50% of new medium?
In general, most cancer cell lines can survive 1 cell/well (dilution or very low density). This is in part due to survival factors cancer cell lines secrete in the medium. Therefore, it may be desirable to provide all survival signals to isolated cells. Use of "conditioned" medium, as you stated, could help. However, one can also add recombinant proteins that are known to increase cell survival in this case. If it turns out that cells do not survive, then other approches can be used to clone cells.
Single cell derived cell generation is a time consuming process irrespective of cells proliferation status. Many cells depend on autocrine factors to accelerate cell division. One way of helping it is, adding less media in 96 well plate wells (not too less: it doesn't dry out) so that secreted growth factors reach good concentration sooner.
Hello Daniil,
Offcourse it's possible to seed a single cell/well in a 96-well plate by serial dilution method (that you must be knowing).
Coming to the media part so I will not recommend you to use old media or condition media derived from other cells or same cells because in case of single cell that old media may not be fully cell free and the centrifugation to viable limits may not be able to pellet down all cells and in turn a contamination of cell/s from previous culture.
I would recommend to use a media with supplementation of growth factors and good quality FBS. It will be sufficient to support the growth of a cell as it's origin is from a cell line.
A single cell will not be able to support it's growth on it's own as area of a well is pretty big and autocrine signal thus may not be available to cell itself thus using a complete supplemented media will be a good option.
Hope it helps..
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