Secondary Assay for Inhibitors of Human Galactokinase (GALK): HEK-293 Cell Viability Assay
Assay Submitter (PI): Kent Lai (University of Utah School of Medicine, 50 N Mario Capecchi Drive, Salt Lake City, UT 84132) ..more
BioActive Compounds: 19
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: R03 MH085689-01
Assay Submitter (PI): Kent Lai (University of Utah School of Medicine, 50 N Mario Capecchi Drive, Salt Lake City, UT 84132)
NCGC Assay Overview
Cytotoxicity of the active compounds in the GALK qHTS screen (AID 1868) was assessed using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, USA) in the HEK-293 (ATCC) cell line. CellTiter-Glo measures the intracellular ATP present in metabolically active cells using Ultra-Glo as the luciferase to detect ATP levels. In brief, the luciferase in the buffer catalyzes the oxidation of luciferin to oxyluciferin and light which is dependent on ATP. Hence, luminescence is proportional to the amount of ATP present in the cell lysis, and this can be used to measure the number of viable HEK-293 cells . Wells treated with various concentrations of cycloheximide (Sigma) and DMSO, and wells with media alone were used as positive and negative controls, respectively. Compounds that do not decrease the luminescence signal are flagged and prioritized.
NCGC Assay Protocol Summary:
HEK-293 cells were grown in a flask up to 80% confluence. Cells were trypsinized, washed, and resuspended in MEM Eagle + 10% FBS + 1% pen-strep media at a concentration of 120 cells/uL. Five uL/well of cell solution was dispensed into a 1536-well assay plates (Greiner, solid white medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Cells were pre-incubated at 37 degree Celsius, 5% CO2 for four hours. Compound and control solutions (23nL) were then transferred to the assay plate using the Kalypsys 1536-pin tool. Assay plates were re- incubated at 37 degree Celsius, 5% CO2. After 24 or 48 hours, five uL of CellTiter Glo detection reagent was added. Luminescence signal was read with Perkin Elmer ViewLux plate reader after 30 min room temperature incubation.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description".
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)