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BioAssay: AID 652042

CEM21 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7013-03_Inhibitor_Dose_CherryPick_Activity

In the fusion assays, a reduction in luciferase signal could be a direct result of cell cytotoxicity and have nothing to do with inhibition of viral fusion. To discard such false positives, the cytotoxicity of each positive hit towards CEM cells will be evaluated using the CellTiter-Glo reagent. Cells will be treated with compounds at multiple doses for 24 hours. Cell viability will be measured using CellTiterGlo (Promega) which measures cellular ATP levels as a surrogate marker of cell viability/proliferation. ..more
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AID: 652042
Data Source: Broad Institute (7013-03_Inhibitor_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-02-15

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 351
Related Experiments
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AIDNameTypeComment
651609Broad Institute MLPCN HIV Entry Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary assay
651610HIV entry: Env-mediated Cell Fusion Measured in Cell-Based System Using Plate Reader - 7013-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
652040Control Cell Fusion Counterscreen Assay Measured in Cell-Based System Using Plate Reader - 7013-02_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
652057HIV entry: Env-mediated Cell Fusion Measured in Cell-Based System Using Plate Reader - 7013-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
652058Cell fusion assay for clade C HIV-1ZM109 Env Measured in Cell-Based System Using Plate Reader - 7013-05_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
652062HIV-1 Cell Fusion assay for clade B Env AD8 Measured in Cell-Based System Using Plate Reader - 7013-04_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
687021Control Cell Fusion Counterscreen Assay Measured in Cell-Based System Using Plate Reader - 7013-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
687023HIV entry: Env-mediated Cell Fusion Measured in Cell-Based System Using Plate Reader - 7013-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
687026CEM21 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7013-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords: CEM, lymphoblast cell line, cytotoxicity, luciferase, CellTiter-Glo

Assay Overview:
In the fusion assays, a reduction in luciferase signal could be a direct result of cell cytotoxicity and have nothing to do with inhibition of viral fusion. To discard such false positives, the cytotoxicity of each positive hit towards CEM cells will be evaluated using the CellTiter-Glo reagent. Cells will be treated with compounds at multiple doses for 24 hours. Cell viability will be measured using CellTiterGlo (Promega) which measures cellular ATP levels as a surrogate marker of cell viability/proliferation.

Expected Outcome: A dose-dependent decrease in luciferase signal will suggest that a compound is cytotoxic and will not be a viable probe candidate.
Protocol
1.Grow CEM21 cells in RPMI induction medium. Split 1:3 or 1:4 for passing.
2.Day 1: Collect CEM21 cells in conical tubes. Spin down the cells at ~1200 rpm for 5 minutes & re-suspend in phenol red free RPMI induction medium (with Puromycin).
3.Count cells, adjust to 3x10;5 cells/mL & seed 3,000 cells per well in 30 uL of RPMI (no Phenol Red) induction medium in Corning 8867BC white 384 well plates.
4.Day 2: Pin transfer 100 nL of compounds to the plate. Incubate the plate 37 degrees C overnight.
5.Day 3: Add 20 uL of room temperature CellTiter-Glo (Promega). Wait 10 minutes & read luminescence with standard luminescence settings on a Perkin-Elmer EnVision plate reader.
Media Recipes
RPMI induction medium (500 mL):
RPMI (-Phenol red) 443 mL
Tet approved HI-FBS 50 mL
Pen-Strep-L-Glu 5 mL
Puromycin (10 mg/mL)50 uL
Notes
.All cells have to be in exponential growth phase. CEM21 cells are split 1:10 every 3 days. Cells should not be allowed to overgrow in the flasks.
.All incubations are done at 37 degrees C in the presence of 5% CO2.
.Cells should be propagated for no more than 30 passages or 3 months whichever is sooner.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE:
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)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
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.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: CEM
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4ACTIVITY_OUTCOME_TEST1The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
5ACTIVITY_SCORE_TEST1The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
6ASSAYDATA_COMMENT_TEST1Comments relevant to the subset of data included in the indicated Test number.String
7AC50_Qualifier_TEST1'>','=', or '<'String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9pAC50_M_TEST1Equal to -1*log10(AC50)String
10Hill_Slope_TEST1The slope at AC50Float
11S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
12Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
16Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
17Activity_at_0.26uM_(%)_TEST1 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.5uM_(%)_TEST1 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_1uM_(%)_TEST1 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_2.1uM_(%)_TEST1 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_4.2uM_(%)_TEST1 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_8uM_(%)_TEST1 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_15uM_(%)_TEST1 (15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_16uM_(%)_TEST1 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_35uM_(%)_TEST1 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
27ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
28ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
29AC50_Qualifier_TEST2'>','=', or '<'String
30AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
31pAC50_M_TEST2Equal to -1*log10(AC50)String
32Hill_Slope_TEST2The slope at AC50Float
33S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
34Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
35Num_Points_TEST2The number of data points used to generate the plotInteger
36Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
37Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
38Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
39Activity_at_0.235uM_(%)_TEST2 (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_0.26uM_(%)_TEST2 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_0.285uM_(%)_TEST2 (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_0.35uM_(%)_TEST2 (0.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_0.5uM_(%)_TEST2 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_0.56uM_(%)_TEST2 (0.56μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_1uM_(%)_TEST2 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
46Activity_at_1.1uM_(%)_TEST2 (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
47Activity_at_1.95uM_(%)_TEST2 (1.95μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
48Activity_at_2.1uM_(%)_TEST2 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
49Activity_at_3.8uM_(%)_TEST2 (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
50Activity_at_4.2uM_(%)_TEST2 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
51Activity_at_4.6uM_(%)_TEST2 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
52Activity_at_7.5uM_(%)_TEST2 (7.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
53Activity_at_8uM_(%)_TEST2 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
54Activity_at_9uM_(%)_TEST2 (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
55Activity_at_15uM_(%)_TEST2 (15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
56Activity_at_16uM_(%)_TEST2 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
57Activity_at_18uM_(%)_TEST2 (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
58Activity_at_30uM_(%)_TEST2 (30μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
59Activity_at_35uM_(%)_TEST2 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 DA034601-01

Data Table (Concise)
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