A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (2)
Project Overview: This functional assay was developed for the detection of compounds inhibiting THP-1 cell viability as a secondary screen to the M. tuberculosis bacteriocidal assay that compares growth in media containing glycerol versus growth in media without glycerol. The THP-1 cell line was chosen as a representative peripheral blood monocyte. ..more
BioActive Compounds: 30
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 the detection of compounds inhibiting THP-1 cell viability as a secondary screen to the M. tuberculosis bacteriocidal assay that compares growth in media containing glycerol versus growth in media without glycerol. The THP-1 cell line was chosen as a representative peripheral blood monocyte.
In this assay, we treated THP-1 cells with compounds selected as "hits" in the comparison with and without glycerol M. tuberculosis assay (AID 449762) for 72 hours over a 10 point 2-fold dilution series, ranging from 40uM to 0.078uM. Following 72 hours of treatment, relative viable cell number was determined using Cell Titer Glo from Promega. Each plate contained 64 replicates of vehicle treated cells which served as negative controls.
Cell Culture: THP-1 cells were subcultured every 7 days in RPMI 1640 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 and carrier controls were diluted in complete growth medium to prepare a 6X concentrated dosing solution which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 uL volume).
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: At the end of the treatment period, assay plates were removed from the incubator and equilibrated to room temperature for 10 min. Twenty-five muL of Cell Titer Glo reagent was added and plates were incubated for an additional 10 min in the dark. At the end of the incubation, assay plates were analyzed using a PerkinElmer Envision microplate reader in luminescence mode 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: THP-1
Assay Type: Functional
Assay Cell Type: NULL
* Activity Concentration. ** Test Concentration.
Data Table (Concise)