I generated a line of HeLa cells which stably express an EGFP plasmid. After going through the process of selecting and growing a colony, the cells were passaged 10 times to ensure their fluorescence was stable. Subsequently, the cells were placed in cryosuspension using cryomedia (10% DMSO, 10% FBS, 80% DMEM) to preserve the population. 24h post-suspension in liquid nitrogen a sample was removed to check cell viability. Despite the cells being viable, they no longer retained their fluorescence and it did not return after a week of growth. This effect was observed among all the cryovials. Has anyone had a similar problem or able to provide any suggestions?
What is the selection antibiotic (G418, Hygromycin, Puromycin...) in the plasmid? Do you add enough antibiotic to retain the plasmid in the cells. Have you checked the dose response to evaluate optimum antibiotic concentration that is required for your plasmid to retain in HeLa cells. Grow your cells in varying concentration of antibiotic and monitor the cell viability and GFP expression for each dose to determine optimum concentration of antibiotic required for your plasmid's stable expression.
Hi Daniel, did you find out why this has happened? This has recently occurred to our lab that our single-cell sorted mCherry-tagged cells lose their fluorescence after cryopreservation. Very frustrating.
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