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BioAssay: AID 652139

HRas Target ID Cytotoxicity multiple timepoints Cell Titer Glo Measured in Cell-Based System

Using CellTiterGlo, which measures the amount of intracellular ATP from living cells, the total amount of viable cells can be measured by luminescence readout. Cells are treated with compounds for for 7 hours, 24 hours or 48 hours with each timepoint having a corresponding assay plate. ..more
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 Tested Compounds
 Tested Compounds
All(7)
 
 
Unspecified(7)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Unspecified(7)
 
 
AID: 652139
Data Source: Broad Institute (2156 HRAS target ID Cell Titer Glo)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-03-19
Hold-until Date: 2014-03-06
Modify Date: 2014-03-06

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
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AIDNameTypeComment
652114Broad Institute MLPCN HRAS target ID Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary
652070HT29 HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-21_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
652072LOX IMVI HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-22_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
652143HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7Othersame project related to Summary assay
652144HRas Target ID Cytotoxicity Multiple Timepoints Alamar BlueOthersame project related to Summary assay
720598HT-29 HRas Target ID Cytotoxicity Multiple Timepoints Cell Titer Glo Measured in Cell-Based System Using Plate Reader - 2156-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720599CCRF CEM HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-19_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720600SNB-19 HRAS Target ID Cytotoxicity Multiple Timepoints Alamar Blue Measured in Cell-Based System Using Plate Reader - 2156-10_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720601C2C12 HRAS Target ID Cytotoxicity Mulitlpe Timepoints Alamar Blue Measured in Cell-Based System Using Plate Reader - 2156-18_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720602LOX IMVI HRas Target ID Cytoxicity Multiple Timepoints Cell Titer Glo Measured in Cell-Based System Using Plate Reader - 2156-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720603C2C12 HRAS Target ID Cytotoxicity Multiple Timepoints Cell Titer Glo Measured in Cell-Based System Using Plate Reader - 2156-17_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720604HUVEC HRas Target ID Cytotoxicity multiple timepoints Cell Titer Glo Measured in Cell-Based System Using Plate Reader - 2156-28_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720605U-251 HRAS Target ID Cytotoxicity Mulitlpe Timepoints Alamar Blue Measured in Cell-Based System Using Plate Reader - 2156-16_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720606LOX IMVI HRas Target ID Cytotoxicity at Multiple Timepoints Alamar Blue Measured in Cell-Based System Using Plate Reader - 2156-08_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720607CCRF CEM HRas Target ID Cytotoxicity multiple timepoints Cell Titer Glo Measured in Cell-Based System Using Plate Reader - 2156-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720608SR HRAS Target ID Cytotoxicity Multiple Timepoints Cell Titer Glo Measured in Cell-Based System Using Plate Reader - 2156-13_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720609HeLa HRas Target ID Cytotoxicity at Multiple Timepoints Cell Titer Glo Measured in Cell-Based System Using Plate Reader - 2156-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720610HeLa HRas Target ID Cytotoxicity at Multiple Timepoints Alamar Blue Measured in Cell-Based System Using Plate Reader - 2156-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720611SNB-19 HRas Target ID Cytotoxicity Multiple Timepoints Cell Titer Glo Measured in Cell-Based System Using Plate Reader - 2156-09_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720612HUVEC HRas Target ID Cytotoxicity multiple timepoints Alamar Blue Measured in Cell-Based System Using Plate Reader - 2156-29_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720613SNB-75 HRAS Target ID Cytotoxicity Multiple Timepoints Alamar Blue Measured in Cell-Based System Using Plate Reader - 2156-12_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720614SNB19 HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-23_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720615U251 HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-26_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720616U-251 HRAS Target ID Cytotoxicity Multiple Timepoints Cell Titer Glo Measured in Cell-Based System Using Plate Reader - 2156-15_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720617CCRF CEM HRas Target ID Cytotoxicity Multiple Timepoints Alamar Blue Measured in Cell-Based System Using Plate Reader - 2156-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720618HeLa HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-20_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720619HT-29 HRas Target ID Cytotoxicity Multiple Timepoints Alamar Blue Measured in Cell-Based System Using Plate Reader - 2156-06_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720624SNB-75 HRAS Target ID Cytotoxicity Mulitple Timepoints Cell Titer Glo Measured in Cell-Based System Using Plate Reader - 2156-11_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720625SR HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-25_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720626SR HRAS Target ID Cytotoxicity Multiple Timepoints Alamar Blue Measured in Cell-Based System Using Plate Reader - 2156-14_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720627C2C12 HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-27_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720629SNB-75 HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-24_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
720630HUVEC HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-30_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords: Ras, apoptosis, cancer, VDAC, oxidative cell death, cytotoxicity, viability, ATP

Assay Overview:
Using CellTiterGlo, which measures the amount of intracellular ATP from living cells, the total amount of viable cells can be measured by luminescence readout. Cells are treated with compounds for for 7 hours, 24 hours or 48 hours with each timepoint having a corresponding assay plate.


Assay: Cell viability post compound treatment via measurement of ATP in:
-CCRF CEM Cells (From ATCC cat # CCL-119: Leukemia, acute lymphoblastic, peripheral blood, T-lymphoblast, CDKN2a Hom c.1_471 del 471 mutant; KRAS Heterozygous c.35G>A, p.G12D; PTEN Hom c. del 80-492; p53 Het c.743G>A, p.R248Q c.524G>A, p.R175H; FLT3 Het c.1879 G>Ap.A627T)
-HeLA Cells (From ATCC: Cervical Cancer derived; low p53 per ATCC, pRB expression normal, other mutations unknown)
-HT-29 Cells (From ATCC Cat # HTB38: Colorectal Adenocarcinoma; APC Het c.2557 G>T p.E853X c.4666_4667 insA; BRAF Het c.1799T>A, p.V600E; SMAD4 Hom c.931C>T, p.Q311X; PIK3CA (GF) Het 1345C>A p.P449T)
-LOX IMVI Cells (From NCI/NCI-60 panel; NCI Cat# 0507374 Malignant Amelonotic Melanoma; CDKN2a Hom c.1_471 del 471; BRAF Het c.1799 T>A, p.V600E)
-SNB-19 Cells (From NCI/NCI-60 panel CNS-Glioblastoma NCI Cat# 0507420; possibly same cell line as U-251 or at least 80 % homology with U-251 cell line; CDKN2a Hom c.1_471 del 471; PTEN Hom c.723_724insTT p.E242fsX15; TP53 Hom c.818G>A, p.R273H)
-SNB-75 Cells (From NCI/NCI-60 panel; NCI Cat# 0507445 CNS-Astrocytoma; TP53 hom c.772G>A, p.E258K)
-SR Cells (From ATCC: Cat# CRL-2262 Large Cell Immunoblastic Lymphoma Metastatic Site: pleural Effusion; CDKN2a Hom c.1_471 del 471)
-U-251 Cells (From NCI/NCI-60 panel; NCI Cat# 0507413 possibly same cell line as SNB-19 or at least 80 % homology with SNB-19 cell line; CNS - Glioblastoma; CDKN2a Hom c.1_471 del 471; PTEN Hom c.723_724insTT p.E242fsX15 ; TP53 Hom c.818G>A, p.R273H)
-C2C12 Cells (Immortalized Mouse myoblast)
-HUVEC Cells (Human Umbilical Vein Endothelial Cells; Primary Cell Line pool of donors from Lonza.)

Expected Outcome: Compounds that are toxic to these cells will produce a decrease in the luminescence readout.
Panel Information
HRas Target ID Cytotoxicity multiple timepoints Cell Titer Glo Measured in Cell-Based System
    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescription
ActiveInactive
1CCRF-CEM51Human acute lymphocytic leukemia cell line
2HeLA42Human cervical cancer cell line
3HT-2952Human colorectal adenocarcinoma cell line
4LOX-IMVI52Human amelonotic melanoma cell line
5SNB-1932Human glioblastoma cell line
6SNB-7551Human astrocytoma cell line
7SR51Human large cell immunoblastic lymphoma cell line
8U-25143Human glioblastoma cell line
9C2C1241Mouse myoblast cell line
10HUVEC22Human umbilical vein endothelial cell line

§ Panel component ID.
Protocol
Protocol:
Compounds used:
D13 layout of compound plate containing 10 compounds in dose (20 doses : final concentration in well 33 uM with 19 subsequent two fold dilutions):
Probe ML162 SID:104543588
ML162 derivative 1: SID: 134959006 {has PEG chain attached to the phenol- it is an OMe on the benzene ring in ML162 with a primary amine that is Boc (tert butyl carbamate) protected }
ML162 derivative 2 SID: 134959009 {Boc removed and replaced with an ester that is 6 carbon chain and ends with a terminal alkyne (triple bond) This may be used for SILAC since it can be attached with an azide coupling partner and copper (Huisgen cycloaddition aka "click" reaction}
Inactive ML162 analog SID: 99351091 ( ethyl amide )
Probe ML210 SID: 103023280
Inactive Analog ML210 (bis-4-pyridyl derivative) SID: 103023276
RSL3: SID: 124360175
Erastin: SID: 160709311
Pifithrin (a, mu) alpha supposedly condensation product to Beta.
pifithrin alpha (stable?) SID: 160709312
pifithrin mu (p53 inhibitor at mitchonodria) SID: 160709313
Growth Media:
HeLA and C2C12 cells are grown in DMEM with 10% FBS. HUVEC cells require a specialty HUVEC media with additives. All other cells use RPMI 1640 medium from Gibco: catalog number : A10491-01 (With 4.5 g/L D-glucose, 1.5 g/L Sodium Bicarbonate, 1 mM Sodium Pyruvate, 10 mM HEPES and 300 mg/L L-glutamine ), 10% FBS. No antibiotics were used in media


Cell Passage
Suspension cell lines (CCRF-CEM and SR):
1.Cells were received from ATCC, thawed and cultured for a few weeks to allow them to adapt to growth in culture. Culture media is RPMI 1640 with 10% FBS. No penicilin/Streptomycin was used in culture media.
2. As this is a suspension cell line, cultures are maintained by the addition of fresh medium or replacement of medium. Alternatively, cultures can be established by centrifugation with subsequent resuspension.
Medium Renewal: Add fresh medium (20% to 30% by volume) every 2 to 3 days. Cells should be maintained at a density between 2 * 10;5 and 2 * 10;6 cells/mL
All other (adherent) cell lines:
Remove and discard culture medium.
Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37 degrees C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37 degrees C.


Assay Day 1
Suspension cell lines:
1. Harvest the cells by centrifugation and resuspension in media.
2. Add media to adjust the concentration of the cells to 50,000/mL.
3. Plate the cells to 384-well white, solid bottom, tissue culture treated plates using the mutlichannel pipetor transferring 30 uL to each well, for a total of 1,500 cells/well.
4. Incubate the plates overnight in a TC incubator at 95% humidity, 5% CO2, 37 degrees C.
Note: although this is a suspension cell line these cells were incubated overnight to keep on the same schedule as the adherent cell lines.
Adherent cell lines:
1.Harvest the cells by first aspirating the media and rinsing the flask with 10 mL PBS. Aspirate the PBS and add 5 mL trypsin to the flask. Incubate with the trypsin for 5 minutes in the TC hood (22 degrees C).
2. Add 8 mL of media to the trypsin. Dislodge the cells from the bottom of the flask by pipeting. Remove an aliquot of cells for counting.
3. Add media to adjust the concentration of the cells to 50,000 cells/mL.
4. Plate the cells to 384-well white solid bottom plates using the mutlichannel pipetor transferring 30 uL to each well, for a total of 1,500 cells/well.
5. Incubate the plates overnight in a TC incubator at 95% humidity, 5% CO2, 37 degrees C.


Day 2
1. Pin with 100 nL compound with cybio walkup using 100 nL head. Pin a total of three plates, one for each timepoint : 8 hours treatment with compound , 24 horus treatment with compound and 48 hours treatment with compound.
2. Return plates to the Liconic incubator at 95% humidity, 5% CO2, 37 degrees C for incubation until appropriate compound treatment time has been reached.
(ie 8 hour timepoint will be on same day as pinning)


Day 2, 3 and 4
1. Place plates on bench for 20 minutes to warm to room temperature.
2. Meanwhile make up Cell Titer Glo reagent by adding lysis solution to substrate. Allow reagent to warm to room temperature.
3. Add 30 ul of pre-warmed Cell Titer Glo reagent to each well (equal volume of plating media).
4. Shake for two minutes and then leave for an additional 8 minutes at room temperature no shaking.
5. Read plates on envision (luminescence); Mirror: Luminescence (bar code 404) Emission Luminescence (bar code 212)
Comment
Compound score determination:If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)then PUBCHEM_ACTIVITY_SCORE = -10*log10(avg(AC50))
Scores relate to AC in this manner:120 = 1pM, 90 = 1nM, 60 = 1uM, 30 = 1mM, 0 = 1M When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1CCRF-CEM_Outcome1Outcome
2HeLA_Outcome2Outcome
3HT-29_Outcome3Outcome
4LOX-IMVI_Outcome4Outcome
5SNB-19_Outcome5Outcome
6SNB-75_Outcome6Outcome
7SR_Outcome7Outcome
8U-251_Outcome8Outcome
9C2C12_Outcome9Outcome
10HUVEC_Outcome10Outcome
11CCRF-CEM_Score1Integer
12HeLA_Score2Integer
13HT-29_Score3Integer
14LOX-IMVI_Score4Integer
15SNB-19_Score5Integer
16SNB-75_Score6Integer
17SR_Score7Integer
18U-251_Score8Integer
19C2C12_Score9Integer
20HUVEC_Score10Integer
21CCRF-CEM_AC50_Test11FloatμM
22HeLA_AC50_Test12FloatμM
23HT-29_AC50_Test13FloatμM
24LOX-IMVI_AC50_Test14FloatμM
25SNB-19_AC50_Test15FloatμM
26SNB-75_AC50_Test16FloatμM
27SR_AC50_Test17FloatμM
28U-251_AC50_Test18FloatμM
29C2C12_AC50_Test19FloatμM
30HUVEC_AC50_Test110FloatμM
31CCRF-CEM_AC50_Test21FloatμM
32HeLA_AC50_Test22FloatμM
33HT-29_AC50_Test23FloatμM
34LOX-IMVI_AC50_Test24FloatμM
35SNB-19_AC50_Test25FloatμM
36SNB-75_AC50_Test26FloatμM
37SR_AC50_Test27FloatμM
38U-251_AC50_Test28FloatμM
39C2C12_AC50_Test29FloatμM
40HUVEC_AC50_Test210FloatμM
41CCRF-CEM_AC50_Test31FloatμM
42HeLA_AC50_Test32FloatμM
43HT-29_AC50_Test33FloatμM
44LOX-IMVI_AC50_Test34FloatμM
45SNB-19_AC50_Test35FloatμM
46SNB-75_AC50_Test36FloatμM
47SR_AC50_Test37FloatμM
48U-251_AC50_Test38FloatμM
49C2C12_AC50_Test39FloatμM
50HUVEC_AC50_Test310FloatμM
Additional Information
Grant Number: R03 MH084117-01

Classification
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