i work on live caco-2 cells. but mostly when i go for confocal microscopy i have to wash the cells with PBS or HBSS to remove the stains used, due to this my cells gets detached and left very less attached for imaging. i tried using high as well as low cell density also, but it didnt work for me. Can anyone suggest who worked on Caco-2 cells? i uses confocal dishes for microscopy.
Dear Shabi!
You look at this article and protocols, please
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I have not worked with caco-2 cells but I do perform live-cell imaging. Fluorobrite medium is available from ThermoFisher that is supplemented medium without phenol red. I use this supplemented with 0.02% BSA or a low concentration of FBS. This tends to maintain the appropriate osmotic environment and prevent cell detachment during washing/imaging. If you cannot obtain this medium you could try adding the BSA or FBS to the HBSS or PBS you are using.
Hello,
You can directly load your cells with the probe by incubating them in your culture medium. So there is no need to wash your cells with PBS/HBSS prior to the loading phase.
Be also careful to use PBS/HBSS with calcium, as even brief moments without calcium can affect cell adherence.
Finally, have you tried to change your culture plates and use ones from another company?
Good luck
You can wash your cells with medium instead of buffer as already suggested above. Additional comment: keep in mind that the temperature also has a key role here. Your washing media should be prewarmed. Hope it helps.
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