Does any one have any experience of creating a stable cell line using Calu-3 cells?

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Eleanor Martin

Thanks Wassim. Approximately, how long does it take in total to create a stable cell line? Would you be able to point me towards a good protocol?

Hi Wassim, thank you very much for your advice. Just one more thing I am unsure about. I have read that this cell line does not grow well at low cell densities. As long as I don't use a limiting dilution type method involving plating one cell/well of a 96 well plate, should it be possible to select a monoclonal line by plating at lowish densities in a 10 cm dish and picking clones as you suggest? Or is it likely that the cells will also not grow well in these conditions? I'm wondering if I could look into collagen coating or a feeder layer should this be an issue.

Dan Dan Wu

Hello Eleanor, how is your Calu-3 stable cell line? I also want to construct a stable cell line using Calu-3, selected with puromucin. I am afraid the cells are difficult to confluencegrow after puromycin selection. do you have some suggestions? thank you. y

Hi Dan Wu, I tried to transfection of Calu-3 using FuGene HD and Lonza's nucleofector system. However I always found that the transfection efficiency was insufficient for creation of a stable cell line. I think that this cell line will not grow well at low cell density, so if after puromycin selection there are very few cells remaining it is unlikely they will grow well. Instead I used lentiviral transduction and FAC sorted my cells several times for GFP positive cells. Lentiviral transduction is a lot more efficient so I could make sure there were sufficient cells to ensure good growth after sorting. However this method may not be appropriate if you need a clonal cell line.

Hi, Eleanor, thank you very much for your suggestion. I also use lentivirus transduction, I don't have GFP tag, so I selected with puromycin. Interestingly, the transduced cells grow very well after puromycin selection, but untransduced cells all died. so I don't know the reason is its high transduction efficiency or the cells protect each other...