Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.
Gap junctions have been suggested as drug targets. Iodide-YFP-GJIC assay is compatible with high-throughput screening to discover gap junction modulators. It have be used to test the effects of a large number of compounds on gap junction activities in a relatively short period.
Iodide-YFP-GJIC assay is simple, robust, repeatable and inexpensive. This assay requires only acceptor and donor cells and two balanced salt solutions. No additional reagents such as Lucifer yellow and Calcein AM are needed.
Also it measures total gap junction activities of the cells in a single well which results in low between-well variability. To begin this procedure grow LN215 cells to 80%confluency in supplemented DMEM as outlined in the text protocol. One day before transduction wash the cells twice with 10 milliliters of PBS.
Add two milliliters of 0.25%trypsin EDTA to the cells and incubate at 37 degrees Celsius for three minutes. Using a 10-milliliter serological pipette resuspend the cells in five milliliters of culture medium and adjust the cell density to 50, 000 cells per milliliter. Add 400 microliters of media which contains 20, 000 cells to each well of a 24-well culture plate.
Incubate at 37 degrees Celsius for 24 hours. The next day transduce two wells by replacing the culture medium with 400 microliters of a one-to-one mixture of a lentivirus and fresh culture medium supplemented with polybrene at a final concentration of four micrograms per milliliter. For the no virus controls replace the culture medium with fresh culture medium supplemented with polybrene at a final concentration of four micrograms per milliliter.
Incubate at 37 degrees Celsius for 15 hours. Then aspirate the medium containing lentiviruses, and fresh culture medium. Incubate the cells at 37 degrees Celsius for an additional 72 hours.
After this, wash the cells in each well twice with 0.5 milliliters of PBS. Add 300 microliters of 0.25%trypsin EDTA to the cells, and incubate at 37 degrees Celsius for three minutes. Resuspend the cells in two milliliters of culture medium, and then plate them in six-well culture plates with two micrograms per milliliter of puromycin.
Culture the cells until all of the cells in the control wells are dead, which usually takes approximately a week. First, separately culture LN215-YFP and LN215-iodide cells in 100-millimeter plates in culture medium to reach the populations required for the assay. One day before the assay, wash each plate with 10 milliliters of PBS.
Treat each plate with two milliliters of 0.25%trypsin EDTA solution, and incubate at 37 degrees Celsius for five minutes. Resuspend the cells in four milliliters of culture medium and transfer them to a 15-milliliter conical tubes. Centrifuge at 1, 000 g for three minutes to pellet the cells.
Discard the supernatant and resuspend each cell pellet in five milliliters of culture medium. Pipette up and down about 20 times to break up any cell clumps. Use a hemocytometer to count the cells.
And then dilute the cells in culture medium to make cell suspension at the densities shown here. Next, mix seven milliliters of each cell suspension together in a reservoir. Use a multichannel pipette to add 100 microliters of this mixture to each well of a 96-well culture plate.
Incubate at 37 degrees Celsius with 5%carbon dioxide for 24 hours. First, use a fluorescence microscope with 20X magnification and a GFP filter to check the 96-well plates to be sure there are no clumps of cells, and that the cultures are fully confluent and well distributed. At least 30 minutes before the assay turn on a microplate reader and set it to 37 degrees Celsius.
Wash the tubing of an automated injector with three milliliters of 70%ethanol, three milliliters of distilled water, and three milliliters of I-solution. Next, warm the C and I-solutions to 37 degrees Celsius in a water bath. Either aspirate the growth medium or invert the plate to empty it.
Tap out any residual medium. Then add the C-solution to a reservoir using a multichannel pipette to add 200 microliters of C-solution to each well of the plate. Either aspirate the solution or invert the plate to empty it, making to tap out any residual solution.
Add 50 microliters of C-solution to each well. Then add one microliter of either a 2.5-millimolar chemical stock or DMSO as a vehicle, and add another 50 microliters of C-solution to each well. Incubate the cells at 37 degrees Celsius in air for approximately 10 minutes.
During the incubation, begin setting the parameters in the microplate reader program by clicking the Manage Protocols button. Click the New button. Select the Fluorescence Intensity in the Measurement Method section, and Well Mode in the Reading Mode section.
Next, click the OK button. A new tab will appear. In the Basic Parameters menu set the Excitation wavelength to 485 nanometers, and the Emission to 520 nanometers.
Select the Bottom optic to read fluorescence from the bottom. Then set the measurement start time to be zero seconds, the number of intervals to be 25, the number of flashes per well and interval to be 20, and the interval time to be 0.4 seconds. In the Layout menu draw a region of the plate to be read.
In the Concentrations/Volumes/Shacking menu set the microplate reader to inject 100 microliters of I-solution to each well with an injection speed of 135 microliters per second. In the Injection Time menu set the injection start time to be one second. Then click the Start measurement button.
When the incubation is complete, transfer the 96-well plates to the microplate reader and start the measurement by clicking the Start measurement button again. In this study, the iodine-yellow fluorescent protein-gap junction-intercellular communication assay is used with LN215-YFP and LN215-iodine cells to screen 2, 320 chemicals to identify novel gap junction intracellular communication modulators. Homosalate is seen to inhibit 50%of gap junction intracellular communication.
While terbinafine completely inhibits it. This confirms terbinafine as a gap junction inhibitor. This assay measures GJIC between neighboring donor and acceptor cells, so the donor and acceptor cells must be completely dissociated before plating.
And the confluence of the mixed culture should be 100%to maximize formation of gap junctions between cells. Time course data, dose response relationships, and assessing the reversibility of gap junction modulators can be obtained with Iodide-YFP-GJIC assay. Also, Iodide-YFP specific connexin GJIC assays can be established by inducing the expression of a connexin of interest into the cells devoid of functional gap junctions.
This makes it possible to determine IC50 value of a chemical of a specific type of connexin. Iodide-YFP-GJIC assay can be used for high-throughput screening, because it is robust, repeatable, and inexpensive. This assays helps to find gap junction modulating chemicals for drug discovery and toxicological assessment.
