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BioAssay: AID 743247

Inhibition of Alexa 488-Fibronectin deposition #on AH1F cell monolayers Measured in Cell-Based System Using Plate Reader - 7059-01_Inhibitor_SinglePoint_HTS_Activity

The deposition of Fibronectin (FN) is well known to be cell-dependent, as molecules at the cell surface serve to bind and arrange FN into a fibrillar mesh. This primary screening assay for identification of inhibitors of FN fibrillogenesis is fibroblast-based, as this is a cell type known to deposit large quantities of FN into the extracellular matrix (ECM). The assay measures fluorescence more ..
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 Tested Compounds
 Tested Compounds
All(342860)
 
 
Active(297)
 
 
Inactive(342567)
 
 
 Tested Substances
 Tested Substances
All(345385)
 
 
Active(297)
 
 
Inactive(345088)
 
 
 Related BioAssays
 Related BioAssays
AID: 743247
Data Source: Broad Institute (7059-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2014-01-08
Modify Date: 2014-01-13

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 297
Related Experiments
AIDNameTypeComment
743248Broad Institute Inhibition of Alexa 488-Fibronectin deposition #on AH1F cell monolayers Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary assay
1035475Inhibition of Alexa 488-Fibronectin deposition #on AH1F cell monolayers Measured in Cell-Based System Using Plate Reader - 7059-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
1053117HEK293 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-01_Inhibitor_Dose_CherryPick_Activity_Set5Confirmatorysame project related to Summary assay
1053118HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_CherryPick_Activity_Set5Confirmatorysame project related to Summary assay
1053119A549 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-06_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords: AH1F cells, HBSS, PBS, fluorescence, FN, ECM


Assay Overview:
The deposition of Fibronectin (FN) is well known to be cell-dependent, as molecules at the cell surface serve to bind and arrange FN into a fibrillar mesh. This primary screening assay for identification of inhibitors of FN fibrillogenesis is fibroblast-based, as this is a cell type known to deposit large quantities of FN into the extracellular matrix (ECM). The assay measures fluorescence intensity from added Al488-FN, which has been ascertained binds and incorporates into the ECM, accumulating in a dose and a cellnumber dependent manner. AH1F cells are plated at 10000 cells/per well in a 384 well plate and allowed to adhere for 1hour. Alexa488 labelled FN is added to the cells and they are allowed to incubate overnight. On day 2 of the assay the plates are washed with Hank's buffered saline solution (HBSS) and phosphate buffered saline (PBS) is added to them. The plates are read for Fluorescence using the envision plate reader.

Expected Outcome:
Compounds that show a 40% or more decrease in fluorescent signal are considered hits and will be subjected to confirmatory and counter screens.
Protocol
1. AH1F cells are plated at 10000cells per well and allowed to adhere for 1hour 30ul volume.
2. Compounds are pinned.
3. Using combi 5ul of 7X (168nM) solution of Alexa 488-Fibronectin is added to the wells to final concentration of 24nM in DMEM Media with 2% serum is added to all the wells. Positive control wells did not receive labeled FN.
4. Cells are incubated overnight
5. Plates are washed 3 times with HBSS
6. 25ul of PBS is added
7. Plates are read using envision excitation 485nM/ emission 525
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Assay Pattern (additive)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -30%.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 50.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid# Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_12.62uM_(%) (12.62μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1 R21 NS067647-01A1

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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