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BioAssay: AID 588702

Luminescence-based toxicity of Human Mammary Epithelial (HMLE) cells_2058-08_Inhibitor_Dose_DryPowder_Activity

Assay Overview: The objective of the experiment is to isolate compounds that are toxic to the background control HMLE cell line. The HMLE cell line expressing shRNA against GFP (HMLE_shGFP) is used as a counterscreen line to identify grossly toxic compounds in HMLE_shECad cell lines (AID 2721). One thousand HMLE_shGFP are plated in 50 uL of media in each well and cells are allowed to adhere more ..
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 Tested Compounds
 Tested Compounds
All(7)
 
 
Active(5)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Active(5)
 
 
Inactive(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 588702
Data Source: Broad Institute (2058-08_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2011-10-26
Hold-until Date: 2012-01-28
Modify Date: 2012-01-28

Data Table ( Complete ):           Active    All
BioActive Compounds: 5
Depositor Specified Assays
AIDNameTypeComment
2721Summary of Broad Institute MLPCN Breast Cancer Stem Cell Toxicity ProjectsummarySummary of Broad Institute MLPCN Breast Cancer Stem Cell Toxicity Project
Description:
Keywords: Breast Cancer Stem Cells, HMLE, toxicity

Assay Overview: The objective of the experiment is to isolate compounds that are toxic to the background control HMLE cell line. The HMLE cell line expressing shRNA against GFP (HMLE_shGFP) is used as a counterscreen line to identify grossly toxic compounds in HMLE_shECad cell lines (AID 2721). One thousand HMLE_shGFP are plated in 50 uL of media in each well and cells are allowed to adhere overnight. The next day, 100 nl compound are added to the wells and are incubated ~72 hours. Cell viability was measured using CellTiter-Glo and luminescence is measured by the EnVision. The chemical compoundsthat are toxic to this cell line would be excluded for further study.

Expected Outcome: Compounds significantly suppressing luminescence, and therefore toxic to HMLE_shGFP will be identified as hits in the screen.
Protocol

Protocol:
CSC media complete media + serum= Propagation media

Using already filtered/sterile components, add:

440 ml DMEM (Cellgro 10-013-CM)
50 ml FBS (HyClone SH30071.03)
5 ml Pen/Strep
5 ml Glutamax-1 (Invitrogen 35050-061)
700ul 50 uM Hydrocortisone (Sigma H6909)
600 ul 10mg/ml Insulin (Sigma I9278)
500 ul 50 mg/ml Gentamicin (Sigma G1397)
250 ul 25 mg/ml Plasmocin (Invivogen ant-mpt)
50 ul 100 ug/ml EGF (Sigma E9644) resuspended in 2% serum/PBS
+
500 ml MEGM Complete Medium (Lonza CC-3051) or MEGM Bullet Kit with components CC-3150
1 ml BPE (Lonza CC-4009)
____________________
Makes 1 liter


CSC media complete media - serum= Screening media

Using already filtered/sterile components, add:

490 ml DMEM (Cellgro 10-013-CM)
5 ml Pen/Strep
5 ml Glutamax-1 (Invitrogen 35050-061)
700ul 50 uM Hydrocortisone (Sigma H6909)
600 ul 10mg/ml Insulin (Sigma I9278)
500 ul 50 mg/ml Gentamicin (Sigma G1397)
250 ul 25 mg/ml Plasmocin (Invivogen ant-mpt)
50 ul 100 ug/ml EGF (Sigma E9644) resuspended in 2% serum/PBS
+
500 ml MEGM Complete Medium (Lonza CC-3051) or MEGM Bullet Kit with components CC-3150
1 ml BPE (Lonza CC-4009)
____________________
Makes 1 liter


HMLE_shGFP cells were previously described in Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell. 2009 Aug 21;138(4):645-59. Epub 2009 Aug 13. PMID: 19682730).

Cells were propagated in Propagation media. For experiments, cells were plated using Screening media.

Cells were seeded in 40 ul of screening medium containing 1000 cells per well into white 384-well opaque-bottom plates (Nunc, Rochester, NY) using an automated plate filler (Bio-Tek uFiller; Winsooki, VT). At 24 hr, 100 nL of compound solutions were pin transferred from stock 384-well plates into the 384-well assay plates containing cells, resulting in 0.010-032 uM final concentration. Cells were incubated for 72 hrs.

HMLE_shEcad lines were each screened in two replicates. Two kinds of negative control wells were employed for normalization: multiple DMSO-only control wells were present on each compound assay plate screened. CellTiter-Glo Reagent (Promega) was added 3 days after compound addition (20 ul/well). Luminescence signal was measured with an automated plate reader (Perkin-Elmer Envision 1).
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1AC50_uM*the concentration whereupon activity reaches 50% of the maximumFloatμM

* Activity Concentration.
Additional Information
Grant Number: 1R03MH089663-01

Data Table (Concise)
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