Table 2.

Calcium Mobilization Assay

Step	Parameter	Value	Description
1	Cells	100 μL	100,000 cells/well, overnight
2	Removing the media
3	Loading buffer	190 μL	2.5 mM probenecid in loading buffer
4	Incubation time	30 min	37°C incubator
5	Compound	10 μL	5% DMSO in HBSS/20 mM HEPES
6	Incubation time	30 min	37°C incubator
7	Baseline reading	16 s	Keep constant temperature in FlexStation (37°C)
8	UII	50 μL	Urotensin II in assay buffer
9	Reading	1 min	FlexStation II
10	Analysis

Step Notes

1. Performed in 96-well clear-bottom plates (Greiner, #655090, coated with poly-l-lysine and laminin). Day 1: cell seeding in DMEM with 10% FBS (UT receptor transfected-HEK293, 100,000 cells/well), 37°C incubator overnight.

2. Day 2: remove the supernatant.

3. Add 190 μL of loading buffer (diluted the Component A with 2.5 mM probenecid in 1 × HBSS/20 mM HEPES) to each well.

4. Incubate cell plate for 30 min at 37°C.

5. Add 10 μL of compounds to each wells. Columns 1–2, 5% DMSO only; columns 3–12, various concentrations of compounds.

6. Incubate cell plate for 30 min at 37°C. Transfer the assay plate to the FlexStation II (using SoftMax Pro) assay plate carriage and run.

7. Baseline measurement. The counter settings were excitation at 485 nm and emission at 525 nm.

8. After 16 s, quickly add 50 μL of urotensin II (×5) by using dispenser in FlexStation II.

9. Read UII-induced calcium signal for 1 min.

10. Analysis respective EC₅₀ or IC₅₀ values.