A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (3)
Project Overview: This functional assay was developed for detection of compounds inhibiting HepG2 cells viability as a secondary screen to the M. tuberculosis bacteriocidal assay that compares growth in media containing glycerol versus growth in media without glycerol (AID 449762). The HepG2 line was selected for cytotoxicity studies since it is derived from a human liver fibroblast and would be more indicative of the type of cell that M. tuberculosis would infect and be applicable as a drug target. ..more
BioActive Compounds: 8
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: William Bishai, Johns Hopkins University
Award: 1 R03 MH084877-01A1
Project Overview: This functional assay was developed for detection of compounds inhibiting HepG2 cells viability as a secondary screen to the M. tuberculosis bacteriocidal assay that compares growth in media containing glycerol versus growth in media without glycerol (AID 449762). The HepG2 line was selected for cytotoxicity studies since it is derived from a human liver fibroblast and would be more indicative of the type of cell that M. tuberculosis would infect and be applicable as a drug target.
In this assay, HepG2 cells were treated with compounds selected as "hits" in the M. tuberculosis assay for 72 hours over a 10 point 2-fold dilution series, ranging from 20 uM to 0.078 microM. tuberculosis 0.39 uM. Following 72 hour incubation period, relative viable cell number was determined using Cell Titer Glo (Promega). Each plate contained 64 replicates of vehicle treated cells which served as negative controls.
Cell Culture: HepG2 cells were subcultured every 7 days in E-MEM with 10% fetal bovine serum, incubated at 37 degrees C in 5% carbon dioxide. Cells were passaged as needed, harvested from flasks using 0.25% trypsin-EDTA and maintained for no more than 20 passages.
Compound Dosing/Plating: Compounds or carrier control (DMSO) were diluted to 6X in complete growth medium and 5 microL was dispensed into 384-well black clear-bottom tissue culture treated plates.
Cell Plating: Twenty uL of complete growth medium containing 3000 cells were dispensed per well. Plates were incubated at 37 C, 5% CO2 for 72h prior to endpoint detection.
Endpoint/Detection: Following the 72 hour incubation period, the assay plates were equilibrated to room temperature for 10 min and twenty-five muL of Cell Titer Glo reagent (Promega) was added to each well using a WellMate (Matrix, Hudson, NH) and the plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a Perkin Elmer Envision microplate reader with an integration time of 0.1 s.
Data Analysis: Sixty-four control wells containing cells treated with DMSO vehicle were included on each assay plate. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100*(Cmpd Lum-Med background)/(Med Cell Ctrl - Med background). The normalized % viability was plotted against the tested concentrations. The CC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0.
Outcome: Compounds that showed <70% cell viability for at least one concentration were defined as "Active". If the % viability at all doses was >70%, the compound was defined as "Inactive".
Scoring: SRBCSC uses a three-tier scoring system where scores of 0-40 apply to primary screen data, 41-80 indicates confirmatory screen data, and 81-100 is reserved for confirmatory data on resynthesized compounds. Inactive compounds at any screening level receive a score of 0. In this confirmatory screen, "Active" compounds were scored based on CC50 results on a tier of 81-100 with "Inactive" compounds scoring 0.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: HEPG2
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)