Fluorescent HTS Cytotoxicity/Cell viability assay (HT1080 cells)
This assay was developed to determine the cytotoxic effects of small molecule compounds on HT1080 fibrosarcoma cells that have been stably transfected with the luciferase gene under the control of the MT1-MMP promoter. This assay is part of the multiplexed assay AID:618, Luminescent HTS for small molecule inhibitors of MT1-MMP transcription. ..more
BioActive Compounds: 364
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Number: XO1 MH78949-01
Assay Provider: Dr. Alex Strongin, Sanford-Burnham Medical Research Institute
This assay was developed to determine the cytotoxic effects of small molecule compounds on HT1080 fibrosarcoma cells that have been stably transfected with the luciferase gene under the control of the MT1-MMP promoter. This assay is part of the multiplexed assay AID:618, Luminescent HTS for small molecule inhibitors of MT1-MMP transcription.
Cytotoxicity/Cell viability is measured using Alamar Blue (resazurin) that detects respiratory activity of live proliferating cells. The generation of the reduced product, resorufin, was detected in fluorescence mode.
This assay was developed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN).
1) HT1080 cells that had been stably transfected with the 7.2 kb 5#-flanking region of the MT1-MMP gene linked to the luciferase gene in the pGL3-Basic vector (Promega), were seeded into Greiner Black, clear bottom plates (cat #781091). 7500 cells were seeded/well in 45ul of DMEM w/o phenol red supplemented with 10% FBS, 1mM Sodium Pyruvate, 2mM glutamine and 20ug/ml gentamicin.
2) Cells are left to adhere overnight at 37 Celsius degrees in a humid environment in the presence of 5% CO2.
3) After the overnight incubation, 5ul of diluted compound (final compound concentration is 10uM, final DMSO concentration is 1%) are added to appropriate wells.
4) 5ul of 10% DMSO are added to negative control wells.
5) 5ul of saturated sodium sulfate are added to positive control wells.
6) The cells are incubated with compound overnight at 37 Celsius degrees in a humid environment in the presence of 5% CO2.
7) After incubation 10ul of 16 fold diluted Alamar Blue (Biosource International DAL1100) are added to each well.
8) The plates are incubated for 4 hrs at 37 Celsius degrees in a humid environment in the presence of 5% CO2.
9) After incubation, the plates are read in fluorescence mode using a Perkin Elmer EnVision, multimode plate reader. The filters used were 531/25nm excitation, 595/60nm emission, and a 555nm dichroic mirror.
Data calculation: Raw data are sent to our biological database (CBIS) whereby % activity of the compounds are calculated based on the intraplate controls of sodium sulfate (positive control) and 1% DMSO (negative control). Compounds that are not cytotoxic will have a low % activity, while cytotoxic compounds will have a high % activity.
For information regarding these cells or the reporter gene construct please contact:
Alex Strongin, Ph.D.
Cell Adhesion & Extracellular Matrix Biology
Sanford-Burnham Medical Research Institute
San Diego, CA 92037
Compounds with greater than 50% activity in reporter gene assay are defined as actives of the primary screening. This assay was a part of multiplexed assay. For dose-response testing, compounds were selected based on the logical conjunction of the following criteria: a) >50% activity in the reporter gene portion of the assay, b) <50% activity in the cytotoxicity assay, c) inactive in AID:411 Luciferase Profiling Assay containing results of MLSCN compound collection partial screening performed by NCGC. These compounds were re-tested in dose-response mode of reporter gene (AID618), cytotoxicity and luciferase (AID 773).
Compounds that show <50% inhibition at 50 uM concentration in the cytotoxicity portion assay were converted to inactive in the outcome column. Compounds with EC50 <50 uM in the cytotoxicity portion assay remain activeindependent of the results of reporter gene or luciferase assays.
Activity scoring rules were devised to take into consideration specific efficacy of a compound and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Activity scoring rules utilized for this assay are as follows:
1. 0-40 scoring range is reserved for primary screen data
a. If primary % Activity is greater than 100%, then the assigned score is 40
b. If primary % Activity is less than 0%, then the assigned score is 0
c. If primary % Activity is between 0% and 100%, then the calculated score is (% Activity)*0.4
2. 41-80 scoring range is reserved for dose response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. For compounds with IC50<50 uM, the score is linearly correlated with a compound#s potency in the cytotoxicity assay using the following equation
c. True positives that have EC50 are assigned a score in the range 50-80 through negative linear correlation with a compound potency:
Score = 44 + 6*(pEC50 - 3)*2.6*(exp(-0.5*nH^2)-exp(-1.5*nH^2)),
where pEC50 is a negative log(10) of the EC50 value expressed in mole/L concentration units and nH is Hill coefficient value. This equation results in the Score values above 50 for well-behaving compounds that demonstrate EC50 < 100 uM.
3. 81-100 scoring range is reserved for resynthesized true positives and their analogues
Categorized Comment - additional comments and annotations
Assay Type: Toxicity
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: HT-1080
* Activity Concentration.
Data Table (Concise)