I am currently working on membrane potential sensitive dyes and I am really struggling with DIBAC4(3). This is the protocol that I have followed by reading the literature. I am using HepG2 cells.
1. seed the cells in a black plate with clear bottom and let attach overnight. the cells are seeded really dense
2. wash the cells with DPBS containing 20mM HEPES 2x to get rid of proteins from the cell culture medium
3. wash once the cells with the corresponding DIBAC concentration (have tried from 4nM to 5000nM)
4. Put the cells in a plate reader adjusted to 37° C (no CO2) and read every 2min for 40min at ex. 485/em 535, cut off 515nm, bottom read.
5. Then I replace the DPBS-HEPES-DIBaC solution by a positive control (have tried 10ug/ml gramicidin and 1000nM valinomycin) in the correspondend DIBAC-solution (this means i add the positive control not directly to the plate but instead prepare the positive control in the DIBAC sol that is used on the cells. according to "molecular devices" this is mandatory to reduce the background).
6. I put the cells back to the plate reader and read again.
7. I see no effects even after half an hour.
my problem is, although valinomycin and gramicidin should work as a positive control, they do not.
can anyone give a useful tipp or sees a problem in the protocol straight away?
thanks a lot,