This protocol enables the recording of a complete functional concentration response relationship of GPCR agonist from one cell population and provides detailed agonist characterization even for low cell sample numbers. The main advantage of serial dosing is the increase in throughput since a single well is sufficient for a full concentration response analysis. This technique is well suited to study single transduction in rare and complex biological models, like organ-on-chip formats in closed microfluidic containers.
The protocol is not difficult to perform, provided the appropriate equipment is available. Preparing the various GPCR ligand solutions at the right concentration is the key to success. Michael Skiba, a graduate student, and Anne Mildner, an undergraduate student, from our laboratory will demonstrate the procedure.
For cell seeding onto the electrode arrays, add the appropriate concentration of cells to the wells of each electrode array, and allow the cells to settle homogenously onto the well bottoms for 10 to 15 minutes at room temperature. After at least 36 hours in a standard cell culture incubator at 37 degrees Celsius and 5%carbon dioxide, inspect the cell layers by phase-contrast microscopy to ensure a complete coverage of each electrode with cells. Then replace the cell culture medium in each well with the appropriate volume of pre-warmed, serum-free medium.
To start an impedance recording, place the electrode array into the connecting array holder of the impedance analyzer, and confirm a proper low impedance contact between the electrodes and the impedance analyzer. Select the electrode type and/or multi-well format in the user interface of the software. If single and multiple frequency data acquisition modes are available and the number of wells to be analyzed is low or the time resolution is not critical, select multiple frequency recordings.
To ensure a maximum time resolution, select a single monitoring frequency. Then start acquisition of the time course data, and select the data folder for where to save the data. The impedance readings will be recorded at the specified number of frequencies for every well To initiate the serial addition protocol in the agonist mode for an eight-well array, add 30 microliters of the lowest concentration of the agonist of interest to the cells.
Then let the cells respond and equilibrate for the predefined period of time before adding 30 microliters of the next highest concentration until each serial dilution of the agonist has been added. To analyze the data, subtract the impedance of the last data point before the first addition of agonist solution, and set the time of addition zero to normalize the impedance values. Plot the time course of normalized impedance, and plot the individual time courses to identify the maxima in impedance after each addition step.
Compose a data sheet with these values, and plot the values of maximum impedance change as a function of the agonist concentration. Then use a data-fitting routine to determine the half-maximal effective concentrations and maximum response using the four-parameter logistic model. In this representative experiment, 10 solutions with increasing histamine concentrations were sequentially added to the cells every 15 minutes, and the change in impedance was measured at each time point.
The impedance changes could then be plotted as a function of histamine concentration and the transfer function of a four-parameter logistic model fitted to the experimental data points from seven wells. To account for possible histamine receptor downregulation, cell desensitization, or overstimulation, the data range for the analysis can be reduced, as shown for this single serial dosing experiment. Three-parameter optimization can then be applied to the data, providing a half-maximal effective concentration of 0.75 plus or minus 0.12 micromolar for this analysis.
The addition of the histamine receptor antagonist diphenhydramine 20 minutes before the first histamine results in a delayed increase of impedance within the serial dosing scheme, corresponding to the need for a higher agonist to elicit a cell response. In the dose response relationship, the effect of the antagonist is expressed as a rightward shift of the curves, corresponding to an increase in half-maximal effective concentration, provided the antagonist is a competitive ligand relative to the agonist. Successful application of the serial dosing scheme is technically easy but requires thoughtful planning, as it is challenging to perform all of the steps with the correct timing.
Serial dosing has paved the way for performing concentration response studies with other readout systems that are limited with respect to the number of samples monitored in parallel. The particular advantages of serial dosing allow the study of receptor-mediated signal transduction with completely new readouts that are sensitive to other phenotypic changes, like cytomechanics. Please wear gloves and goggles when preparing the stock solutions of the receptor agonist, as they may be hazardous when inhaled or swallowed.
