My protocol requires the establishment of clonal cell lines through the expansion of single cell clones sorted in individual wells by FACS. This is notoriously challenging for fibroblast cell lines such as BJ, which often do not proliferate as isolated cells. Do you have tips for stimulating fibroblast cell proliferation after single-cell sorting?
When I have need to clone in a 96 well format, I used the recommended basal media with 20% FBS and 2x the usual amount of glutamine. After verifying which wells have single cells, I let them incubate for at least a week without movement as to not allow endogenous growth factors to disperse. Any feeding is done by replacing half the media with cloning media. As they sit for prolonged times without media change, I would not seed the edge wells, but would fill them with sterile water. It might help to use collagen I coated plates. Another strategy would be to use conditioned media (24hr growth media from 70-80% confluent cells, centrifuged 1200 rpm 10 min, 4C, then filtered through a .2 um filter) from uncloned fibroblasts, though you would need to titrated the ratio of conditioned media to cloning media, not exceeding 30-40%. Hope this helps and good luck!
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