Table 1.

Dynamic Mass Redistribution Assays

Step	Parameter	Value	Description
1	Cells	30 μL	30,000 cells/well, overnight
2	Wash	25 μL × 3	Pipetting and aspiration
3	Buffer #2	30 μL	1% DMSO with assay buffer
4	Incubation time	2 h	Equilibrate in EnSpire (25°C)
5	Baseline reading	4 min	Keep temperature constant
6	Compound	10 μL	Compounds in 1% DMSO with assay buffer
7	Final reading #1	30 min	EnSpire (25°C)
8	UII	10 μL	Urotensin II in assay buffer
9	Final reading #2	30 min	EnSpire (25°C)
10	Analysis

Step Notes

1. Performed in EnSpire-LFC, 384-well fibronectin-coated plates. Day 1: cell seeding (UT receptor transfected-HEK293, 30,000 cells/well), 37°C incubator overnight.

2. Day 2: washing with buffer #2 (1 × HBSS, 20 mM HEPES, 1% DMSO), 25 μL × 3 remove all with aspiration.

3. Add 30 μL of buffer #2.

4. Equilibrate for 2 h in EnSpire (keep temperature constant). While cell plate is equilibrating, prepare compound dilution plates.

5. Baseline measurement.

6. Add 10 μL of compounds (×4). Columns 1–3, buffer #2; columns 4–12, various concentrations of compounds.

7. Quickly reload plate into EnSpire for final read #1(∼30 min).

8. Add 10 μL of urotensin II (×5).

9. Quickly reload plate into EnSpire for final read #2(∼30 min).

10. Analysis respective EC₅₀ or IC₅₀ values.