I am interested to grow colonies from individual cells (clonal propagation) in order to isolate pure cell line with modified gene after CRISPR genome editing. Looks like my cell line not able to form colonies from dilution of one cell per well. Does anyone have an idea what other options could be?
Most cell have a threshold cell number below which cell grow poorly. One method I use to improve the chances of obtaining single cell clones is to conditioned media from a healthy rapidly dividing parental cells. I use a 1:1 ratio of fresh media and conditioned media. The condition media contains cell secreted growth factors that improve growth/survival rate of the single cells.
Most cell have a threshold cell number below which cell grow poorly. One method I use to improve the chances of obtaining single cell clones is to conditioned media from a healthy rapidly dividing parental cells. I use a 1:1 ratio of fresh media and conditioned media. The condition media contains cell secreted growth factors that improve growth/survival rate of the single cells.
Thanks Daniel. I'll try conditioned media as you suggested. I just thought to find out what approaches have been tried by others. I have heard that in some cases conditioned media is not enough as something is still missing - maybe cells need physical contact. I am not sure how to tackle the problem.
As Daniel suggested, conditioned medium is the most immediate "best" option here.
Another suggestion is to use a very small volume culture vessel, such as a Terasaki (microtest) plate. The wells are only 20 uL in volume, so are very convenient for isolating single cells.
The paper below describes my protocol for isolating live single cells with subsequent culture by limiting dilution in a Terasaki plate. Instead of going ahead and doing gene expression analysis, though, you would continue to culture them.
Article A convenient, optimized pipeline for isolation, fluorescence...
As an alternative to Daniel's suggestion, i.e. to use conditionned medium, you may use irradiated wild type cells (as "feeder cells").
And if you don't have acces to an irradiator, mitomycine B should work as well.
The "treated" cells will not proliferate and die after a week or so, just the time for your cells of interest to grow. Of course, the optimal X-ray or mitomycine doses will depend on your cell type and should be evaluated beforehand.
I would suggest the feeder cell route (either irradiated or mytoC). If you do this on a larger plate and mark your feeders (any cell membrane dye should work as the cells aren't dividing) then you can identify any colonies of growing cells quite easily.
what about ROCK inhibitor y27632 which can inhibit the signaling between cells.
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