Hi everyone,
Currently I am trying to calcium imaging using HEK293T cells,
however, I can not measure fluorescence because HEK293T cells can easily be stripped after following procedure,
I already tried to use collagen coated plate and zelatin coated plate but both did not work as well.
Could anyone teach me good method if you are familiar with that, thank you so much
My procedure is following
1, seeds HEK293T cells 4×10^4/well to 96 well plate
2, remove culturing medium and wash with PBS.
3, Add loading buffer (recording medium + Fluo4AM probe), incubate at 37℃, 1hr
4, remove loading buffer and wash with PBS
5, Add recording medium
6, Add ionomycin to induce calcium ion inside of the cells
7, measure fluorescence
Hi Shunsuke,
Maybe a stupid question but do you have Ca2+ in your recording buffer? It may be important to have some for the adhesion of your cells.
You can also reduce the time of incubation. In my experience, 30min can be enough to load the cells.
However, you may also consider to include in your procedure, 20-30min without dye after loading to allow the de-esterification of Fluo-4-AM in your cells.
Good luck with your experiments
Best
Benjamin
Have you tried with pluronic acid in the Fluo-4 solution? whether the fluo-4 is AM then you need to add pluronic acid for doing the membrane cell permeable to the probe.
best.
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