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Table 3 qHTS protocol

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

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
  1. Notes
  2. 1 Black clear bottom 1536-well plates, single tip dispense of 2500 cells/well into all wells
  3. 2 37°C, 5% CO2 incubator
  4. 3 Pintool transfer of library to columns 5-48, and controls to columns 1-4.
  5. 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.
  6. 5 37°C, 5% CO2 incubator
  7. 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. 8 460/25 nm and 530/20 nm emission filters