Fluorescence-based membrane potential assays provide robust methods for studying the effects of modulators on ion channels that are endogenously expressed in epithelial cells. As proof of concept, we are measuring the function of two ion channels in well-known epithelial cell lines. This assay is versatile, since it uses an exogenous chemical probe to measure ion channel function, therefore bypassing the need for genetically-encoded probes.
These high-throughput assays can identify and characterize the effects of small molecule modulators on ion channels, which have the potential to advance therapies for cystic fibrosis. To begin, culture Calu-3 and Caco-2 cells in a T75 flask containing EMEM with 20%fetal bovine serum and 1%penicillin streptomycin. Aspirate the medium from the T75 flask after the cells reach 80 to 100%confluency.
Gently wash the cells with 10 milliliters of phosphate-buffered saline. Then, add 2 milliliters of prewarmed 0.25%trypsin, with 0.1%EDTA, to the cell monolayer. Place the flask at 37 degrees Celsius for approximately five minutes.
In this step, ensure that the cells lift from the flask surface and dissociate into the single cell suspension. Add 8 milliliters of culture medium to neutralize the reaction. When the cells reach 30 to 40%confluency, add 1.5 milliliters of the cell suspension to 18.5 milliliters of culture medium in a conical tube and mix.
Add 200 microliters of this cell suspension to each well of a 96-well plate to get approximately 140, 000 cells per well. To plate one full 384-well plate, add 1 milliliter of the cell suspension to 17 milliliters of the culture medium in a conical tube to get approximately 50, 000 cells per well. After mixing, add 50 microliters of the cell suspension to each well.
Change the medium every two days and ensure that the cells reach 100%confluency within three to five days in all wells simultaneously. Change the medium 24 hours prior to the functional studies. First, prepare the sodium-free, chloride-free buffer solution with the reagents listed in the text protocol.
Add the reagents to tissue-culture grade, double-distilled water. Allow the reagents to dissolve by stirring overnight at room temperature. After the solution is stable, adjust the pH to 7.4 by adding 1 molar NMDG solution dropwise.
Mix for 30 minutes and adjust the osmolarity of the solution to a range of 300 plus or minus 5 millimoles per kilogram. Filter and store the buffer solution in sterile bottles. Then, dissolve 0.5 milligrams of the membrane potential dye in 1 milliliter of sodium-free, chloride-free buffer and warm the solution to 37 degrees Celsius.
Remove the culture medium from Calu-3 and Caco-2 cell monolayers. Add 195 microliters of the dye solution per well for the 96-well plate, and 95 microliters per well for a 384-well plate. Allow the cells to load the dye for 35 minutes at 37 degrees Celsius and 5%carbon dioxide.
Then, take fluorescence measurements with excitation at 530 nanometers and emission at 560 nanometers. Initially, take continuous baseline readings for five minutes at 30-second intervals. Then, add 5 microliters of 400 micromolar Forskolin solution to each well of the 96-well plate, to get a final Forskolin concentration of one micromolar.
For the 384-well plate, add five microliters of 20 micromolar Forskolin solution. Take a stimulation rating for 20 minutes with measurements at 15-second intervals. Next, add 5 microliters of a 400 micromolar solution of CFTR inhibitor to the 96-well plate, to get a final concentration of 10 micromolar.
To the 384-well plate, add 5 microliters of 200 micromolar CFTR inhibitor. Take an inhibition reading for 15 minutes with measurements at 30-second intervals. Quantify the fluorescence intensity measurements of each well and export the values as a spreadsheet.
in column format, containing individual wells. To calculate Forskolin-induced changes, divide the relative fluorescence unit measurements from each well of the 96-well plate by the last fluorescence intensity measurement of the baseline reading, and plot them. Measure the peak responses as the maximum fluorescence intensity measured from baseline during Forskolin stimulation.
Use this measurement, or area under the curve, to quantify the CFTR response. CFTR function was detected as membrane depolarization after Forskolin stimulation in Caco-2 cells. Fluoride efflux was detected as a sharp increase in fluorescence compared to DMSO as the vehicle control.
After Forskolin stimulation, the fluorescent signal was sustained until the addition of CFTR inhibitor, after which there was a rapid decline in the fluorescence intensity. This phenomenon was reproducible in both Caco-2 and Calu-3 cells. CFTR activity was calculated as the difference in the maximal change in fluorescence after Forskolin or DMSO stimulation.
Individual points ranged between plus or minus 3 standard deviations from the mean in the case of Forskolin stimulation and the DMSO control, indicating the reproducibility of the assay. To confirm the specificity of functional responses, these membrane potential assays can be further validated using conventional electrophysiological methods, such as Ussing chamber. This platform is able to bridge the gap between high-throughput modulator screens and heterologous expression systems and time-consuming bioelectric measurements in difficult-to-access primary tissues.
