Does one add the high-concentration of KCL (~40-50mM) solution directly into the dish as its being imaged?
Does the calcium-dye loading needed to be done in the above solution as well?
Hi!
I have done thousand of times the experiment you mentioned above. First you dye your cells with your calcium-dye (a period of 20min is reasonable) diluted in your desired non-depolarizing solution. Then you can directly image your cells and stimulate during imaging with the KCl solution.
Good luck!
@Matheus De Castro Fonseca @Florian Hetsch
I am using HBSS for my dye loading. Post-dye loading, I prepare HBSS, where I increase the KCL concentration and decrease NaCl by the same factor.
While imaging, I replace the HBSS medium and take basal recording (no media) and then after few seconds add high-KCL buffer. However, the cells don't look healthy and it takes >100s to reach baseline.
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