Identification of small molecule compounds that inhibit the HIF-1 signaling pathway

Table 3 qHTS protocol

Step	Parameter	Value	Description
1	Plate cells	5 μL	2500 HRE-bla Cells
2	Incubation time	4-6 hr	Cells adhere and acclimate
3	Library Compound Control Compound	23 nL 23 nL	38 μM to 0.5 nM titration series 0.1 nM to 2 μM titrations (NSC 607097)
4	Reagent	1 μL	Buffer +/- agonist
5	Incubation time	17 hrs	Induce HRE reporter gene
6	Reagent	1 μL	β-Lactamase detection mixture
7	Incubation	2.5 hrs	Cells load and cleave dye
8	Assay Readout	Ex = 405/8 nm	Envision

Notes
1 Black clear bottom 1536-well plates, single tip dispense of 2500 cells/well into all wells
2 37°C, 5% CO₂ incubator
3 Pintool transfer of library to columns 5-48, and controls to columns 1-4.
4 Single tip dispense for each reagent, Column 1 and 4-48, agonist-60 μM CoCl2. Column 2, agonist-120 μM CoCl₂. Column 3, buffer only.
5 37°C, 5% CO₂ incubator
6 Single tip dispense of 0.6 μM CCF4-AM, 1 mg/ml Pluronic F127 surfactant, 3.5% PEG 400, 2.6% TR40, 2 mM probenecid, 0.1% DMSO
8 460/25 nm and 530/20 nm emission filters

ISSN: 1476-4598