I have already prepared knock out cell by using px459 for knock out mcf7 cell then shorting by using FACS to get single clone, one day after FACS I get around 10 single clone/well ( positive EGFP signal) in 96 well plate then incubation 1-2 week, but after one week incubation I did not get colonies. My cells did not grow up. Please give any suggestion. What should I do??
Thanks very much for your suggestion
Try to use conditioned medium. A more detailed description can be found here on RG:
https://www.researchgate.net/post/Does_anyone_have_an_idea_how_long_it_will_take_for_a_single_mcf7_cell_to_grow_into_a_clump_or_double_for_that_matter
Try to use conditioned medium. A more detailed description can be found here on RG:
https://www.researchgate.net/post/Does_anyone_have_an_idea_how_long_it_will_take_for_a_single_mcf7_cell_to_grow_into_a_clump_or_double_for_that_matter
In my experience, MCF7 cells grow as epithelial cells and require survival signaling from cell-cell contact to grow.
Personally, I would keep all sorted (GFP-positive) MCF7 cells together, culture them to get a full flask, and then sort again. By keeping all cells together (instead of single cell colonies) you will retain the natural heterogeneity that exists in your cell lines.
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