Table 2.

HTS Assay Protocol

Step	Parameter	96-well plate	384-well plate	Description
1	Plate cells	50 μL	12.5 μL	50,000 cells/well for 96-well plate; 12,500 cells/well for 384-well plate
2	Controls	50 μL	12.5 μL	DMSO, 17-AAG, 174
3	Library compounds	50 μL	12.5 μL	Dilution range 1.2 nM–25 μM
4	Incubation time	1 h	1 h	37°C, 5% CO2
5	Reporter reagent	100 μL	50 μL	Luciferin substrate
6	Assay readout			CCD imager, luminescent mode

Step Notes

1. Prepare drug plates in DMSO by serial dilution of 20 mM drug stocks (dilution range 100 nM–2 mM). Final volume is 30 μL for 96-well plate or 20 μL for 384-well plate.

2. Dilute control and test compounds from drug plates in media to make dilution plates. Final volume is 200 μL for 96-well plate or 80 μL for 384-well plate.

3. Add drug in media from dilution plate to 96-well plate (Costar 96-well white opaque plate) or 384-well plate (Corning 384-well black plate with clear bottom) at the volume indicated earlier.

4. Add heat-treated (50°C) cells to each well of cell plate at the volume and concentration indicated earlier.

5. Incubate plate at 37°C for 1 h.

6. Add reporter reagent (75 mM tricine, pH 7.8, 24 mM MgSO₄, 0.3 mM EDTA, 2 mM DTT, 0.313 mM luciferin, 0.64 mM coenzyme A, 0.66 mM ATP, 150 mM KCl, 10% Triton X-100, 20% glycerol, and 3.5% DMSO) at the volume indicated earlier. Shake plate for 2 min.

7. Read luciferase activity (relative light units, Rel. LU) across plate.