Competitive Binding Assay to Identify Compounds Disrupting Receptor-Ligand Interactions

To begin, dispense 100 microliters of compound solution into a clear 96-well round-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 multichannel pipette. Incubate the plate for 15 minutes at room temperature in the dark.

Following incubation, add 50 microliters of fluorescently-labeled 100 nanogram 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 5 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 assay buffer from a reagent reservoir to the wells using a multichannel pipette.

Immediately proceed to centrifuge the plate again for 5 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-blot format as forward scatter area, side scatter area, and, in a histogram view, the fluorophore detection channel.

Choose one sample, for instance, a negative control sample, to perform gating of a defined homogeneous 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 fluorophore detection will now appear on the screen.

Now, select the software's gating tool. Based on the forward scatter and side scatter dot-blot plot visualization, pre-define a homogeneous 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 gate 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, select "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.