After growing endothelial cells in the 96 well plate, remove it from the incubator and confirm confluency using an inverted microscope. Flick the plate into a sink to remove the complete media, and blot the top of the plate with a paper towel. Add HBSS HEPES assay buffer plus probenecid solution to a 50 milliliter multi-channel pipette solvent reservoir.
Using a multi-channel pipette, add 100 microliters of assay buffer to each well and allow the cells to sit for five minutes. Then, flick the plate to remove the assay buffer. Add downloading buffer to a separate 50 milliliter multi-channel pipette solvent reservoir.
Using a multi-channel pipette, add 50 microliters of the dye-loading buffer to each well of the assay plate. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 45 minutes. After incubation, observe the plate under a fluorescence microscope to confirm the dye uptake.
Flick the plate to remove the dye solution. Wash the cells twice with 50 microliters of the HBSS HEPES assay buffer plus probenecid solution in each well. Flick the plate to remove the assay buffer.
Add 92 microliters of HBSS HEPES assay buffer to each well, and again, view the cells with a fluorescence microscope to ensure excess dye has been fully removed and that cells remain adherent. Using a multi-channel pipette, add two microliters of the antagonist solutions and vehicle to appropriate wells. After turning on the plate reader, set the temperature to 37 degrees Celsius and incubate the plate for 15 minutes.
Measure the background fluorescence in columns one and two, performing five scans per well. Eject the plate from the plate reader, then from an 8-tube PCR strip, quickly add six microliters of agonist solution using a multi-channel pipette to each well in columns one and two. Measure the change in fluorescence as calcium mobilization occurs in the cells.
Perform 20 scans of each well. The calcium mobilization assay with endothelial cells demonstrated that positive control wells with no antagonist exhibited a rapid increase in fluorescence. Wells with high antagonist concentrations showed minimal change in fluorescence, similar to the negative control wells with no agonist.
