Characterizing Modulators of Protease-activated Receptors with a Calcium Mobilization Assay Using a Plate Reader

Abstract

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of Gαq signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

Figure 1:. Fluo-4/AM loaded into EA.hy926 cells.

Fluo-4 selectively loaded into EA.hy926 cells, after dye incubation and washing steps. All fluorescence is within cells, with no dye leakage. Image obtained using the GTP setting on an EVOS cell imager and 20x magnification.

Figure 2:. Potential output of assay.

(A) Representative plate reader output of a working assay. Row A is the negative control and has no change in fluorescence. Row B is the positive control and shows a rapid increase in fluorescence. Rows C-H are the experimental wells, with the higher concentrations of parmodulin lower in the plate. (B) Example output of a failed experiment. No increase in fluorescence is seen in row B, which is the positive control. (C) Change in fluorescence over time induced by the PAR1 agonist TFLLRN-NH₂. Scanning two columns of a 96-well plate using the provided settings takes ~5 min. on the PerkinElmer EnSpire plate reader (i.e., ~15 s between scans of each well). Different NRD-21 solutions are as indicated, “Agonist” is 6.5 μM TFLLRN-NH₂, n = 1.

Figure 3:. Example Excel output for a working assay.

Basal fluorescence is output into even-numbered rows starting at row 10, and the result from the first 10 scans after agonist addition is output shown in subsequent odd-numbered rows.

Figure 4:. Robotic liquid handling vs “flicking.”

Precoating the assay plates with gelatin and manually performing media exchanges by flicking the plate and blotting it dry produces concentration-response curves with lower variability. (A) Concentration-response curves of parmodulins ML161 and NRD-21 and 5 μM TFLLRN-NH₂, n = 3 (automated liquid handling). (B) Concentration-response curves of parmodulins ML161 and NRD-21 and 5 μM TFLLRN-NH₂, n = 3 (gelatin-coated, flicked plates). Data are means ± SEM.

Figure 5:. Temperature studies.

Performing the assay at 37 °C generates more reproducible data than at room temperature. (A) Concentration-response curves of ML161 and 5 μM TFLLRN-NH₂, n = 3 (room temperature). (B) Concentration-response curves of ML161 and 5 μM TFLLRN-NH₂, n = 3 (37 °C). Data are means ± SEM.

Figure 6:. Cell density studies.

The optimal cell density is 60,000 cells/well when seeding 16–24 h prior to beginning an assay. (A) Concentration-response curves of TFLLRN-NH₂ at different cell densities, n = 3. (B) Concentration-response curve of TFLLRN-NH₂ at the optimal cell density of 60,000 cells/well. Data are means ± SEM.

Figure 7:. Assay volume.

Using an assay volume of 100 μL generates more reproducible concentration-response curves than assays using a final volume of 200 μL. (A) Concentration-response curves of ML161 and 5 μM TFLLRN-NH₂, n = 3 (200 μL final volume). (B) Concentration-response curves of ML161 and 5 μM TFLLRN-NH₂, n = 3 (100 μL final volume). Data are means ± SEM.

Figure 8:. Generation of Z’ factors to determine the ability of the assay to detect a hit compound.

Calcium mobilization responses with 6.5 μM TFLLRN-NH₂ (column B) plus optional PAR1 antagonists 10 μM NRD-21 (column C) or 0.316 μM vorapaxar (column D). (A) Assay completed 16 h after cell seeding. (B) Assay completed 24 h after cell seeding. (C) Table of calculated Z’-factors using the following equation: $Z^{\prime }=1-3\left({\sigma }_p+{\sigma }_n\right)/\left|{\mu }_p-{\mu }_n\right|$, where σ_p is the standard deviation of the positive control, σ_n is the standard deviation for the negative control or sample, μ_p is the mean of the positive control, and μ_n is the mean of the negative control or sample. Each value is calculated by designating vehicle + 6.5 μM TFLLRN-NH₂ as the positive control and comparing to the indicated condition as the negative control (vehicle) or sample (NRD-21 and vorapaxar).

Figure 9:. Calcium mobilization from a PAR2 agonist.

Change in fluorescence over time induced by 10 μM SLIGKV-NH₂, n = 4. Data are means ± SEM.