I am running the assay on Caco-2 cells using a micro-plate reader. I used the DiSBAC2(3) dye, with excitation and emission wavelengths of 530 nm and 560 nm. I am seeding the cells into 6-well plate in with 2 ml for each well at concentrations of (5-6 ×105 cells/mL), incubating them until 100% confluency. I remove cell medium, and rine wells twice with DPBS, and then add 2 ml of a solution containing (in mM): 50 sucrose; 5 dextrose; 125 NaCl; 4 KCl; 1 CaCl2; 1 MgCl2; 5 HEPES, then I incubate cells with 500 nM of DiSBAC2(3) for 15 minutes, while being covered in foil. I use the Infinite® 200 Pro microplate, and i heat it to 37oC, the measurements are taken from the bottom. I take an initial reading, then I remove the plate after 5 minutes, add the Forskolin/IBMX mixture, and take one measurement every 5 minutes. I add 10 µM forskolin and 100 µM IBMX, DMSO concentration is 0.2%.