The overall goal of the cell-based Assay is to identify molecules that bind to a G protein coupled receptor. Based on their capability to compete with a fluorescently-labeled ligand for binding to the receptor. Competition binding assay can support G-protein coupled receptor drug discovery.
As we will demonstrate with this protocol for the chemokine receptor, CXC4. The main advantage of this method is that living cells are used to analyze receptive binding instead of memory preparations. And that the use of radioactivity label probes is avoided.
Though this method has initially been set up to study binding to the chemokine receptor CXCR4, it can also be developed for other GPCR for which fluorescently-labeled ligands exist. To begin, dispense 100 microliters of compound solution into a clear 96 well rounded bottom plate, according to a predefined experimental layout. Add 50 microliters of cell suspension from a reagent reservoir into the 96 well plate using a multi-channel pipette.
Incubate the plate for 15 minutes at room temperature in the dark. Following incubation, add 50 microliters of fluorescently-labeled 100 nanograms per milliliter CXCL12 from a similar reagent reservoir to the wells of the 96 well-plate. Incubate for 30 minutes at room temperature in the dark.
Next, centrifuge the 96 well plate at 400 times gravity for five minutes at room temperature. Remove the supernatant from the pelleted cells by flipping over the plate. Dry the plate on a tissue.
Add 200 microliters of fresh SA buffer from a reagent reservoir to the wells using a multi-channel pipette. Immediately proceed to centrifuge the plate again, for five minutes at 400 times gravity at room temperature. Again remove the supernatant, by flipping over the plate and then drying it on a tissue.
Finally, gently resuspend the cell pellet in 200 microliters of 1%paraformaldehyde dissolved in PBS. This step will fix the cells. To analyze the samples by flow cytometry, start up the device and open the corresponding software.
Select the cellular parameters to be visualized in a dot block format, as forward scatter area, side scatter area, and in a histogram view, the fluoro four detection channel. Choose one sample. For instance a negative control sample, to perform gating of a defined homogenous cell population based on the forward scatter and side scatter parameters.
Adapt the settings for loading the cells into the flow cytometry device. Select three mixes before injection. Select automatic injection of 100 microliters of fixated cells, and use a sample flow rate of 1.5 microliters per second.
Run the sample by selecting acquire data. The forward scatter and side scatter parameters for this sample, as well as the fluoro four detection will now appear on the screen. Now, select the software gating tool.
Based on the forward scatter and side scatter dot plot visualization, predefine a homogenous and viable cell population by gating. Select to analyze 20, 000 events per sample. This means that for each sample, 20, 000 cells that fall within a predefined gates, will eventually be analyzed.
Data acquisition will continue until this number of events is reached. Start the run by selecting first, the wells to be analyzed. Then selects run wells.
The flow cytometry device will now analyze these samples one by one, by recording the mean fluorescence intensity for each sample. Which corresponds to the mean fluorescent signal from the 20, 000 cells that fall within the predefined gate. Finally, perform data analysis as described in the protocol.
Following pre-incubation of jurkat cells with high concentrations of AMD3100, unestablished and specific CXCR4 antagonist. The mean fluorescence intensity that reflects binding of labeled CXCL12 is almost entirely reduced to background levels. This demonstrates the highly specific binding of CXCL12 to CXCR4 expressed on jurkat cells.
Compounds that are unable to compete with labeled CXCL12 for binding at the CXCR4 receptor, like the CCR5 antagonist Maraviroc, will not affect the mean fluorescence intensity. In contrast, highly active compounds will do this in a dose-dependent manner. After watching the video, you should have a good understanding of how to perform a competition receptive binding assay using living cells and flow cytometry.
