Bookmark and Share
BioAssay: AID 588810

HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_Activity_Set2

HDL binding is assessed using Alexa488 labeled HDL particles. The assay is similar to the primary DiI-HDL screening assay, except DiI non-specifically enters lipid membranes both on HDL particles and cell membranes. For this assay, the Alexa488 is directly bound to apolipoprotein components of the HDL particle and so no transfer of the fluorophore to cell membranes occurs. Instead, direct binding of the HDL particles to the SR-BI receptor can be measured. Compounds will be tested at dose. As a positive control, BLT-1 which increases binding will be used at 1 uM. ..more
_
   
 Tested Compounds
 Tested Compounds
All(34)
 
 
Active(32)
 
 
Inactive(1)
 
 
Inconclusive(1)
 
 
 Tested Substances
 Tested Substances
All(35)
 
 
Active(33)
 
 
Inactive(1)
 
 
Inconclusive(1)
 
 
AID: 588810
Data Source: Broad Institute (2085-03_Inhibitor_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-11-17
Hold-until Date: 2012-11-10
Modify Date: 2012-11-13

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 32
Depositor Specified Assays
AIDNameTypeComment
488952Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe ProjectsummarySummary assay
Description:
Keywords: HDL binding, SR-BI, fluorescence, cell-based assay


Assay Overview:
HDL binding is assessed using Alexa488 labeled HDL particles. The assay is similar to the primary DiI-HDL screening assay, except DiI non-specifically enters lipid membranes both on HDL particles and cell membranes. For this assay, the Alexa488 is directly bound to apolipoprotein components of the HDL particle and so no transfer of the fluorophore to cell membranes occurs. Instead, direct binding of the HDL particles to the SR-BI receptor can be measured. Compounds will be tested at dose. As a positive control, BLT-1 which increases binding will be used at 1 uM.

Expected Outcome: It is possible that a compound can reduce binding of HDL to the receptor and this would lead to a decrease in signal. To date, most compounds, like BLT-1, seem to increase binding to the receptor leading to an increase in signal. This assay is used to decipher the mechanism of activity for a particular compound. Therefore, any outcome in the assay is acceptable.
Protocol

ldlA[mSR-BI] Chinese Hamster Ovary (CHO) cells are maintained in Ham's F12K (Cellgro Catalog # 10-080-CV)/10% Fetal Calf Serum (Hyclone Catalog # SH30071.03, lot # ATG32533)/ 1x Penicillin-streptomycin-L-Glutamine (GIbco 10378-016)/200 ug/mL Geniticin (Gibco Catalog #10131-027, lot#802967). Cells are fluid changed every 2 days and/or split upon reaching 80% confluency.

On day 0, 10,000 ldlA[mSR-BI] cells were plated in 30 microl per well in Ham's F12K (Cellgro Catalog # 10-080-CV)/5% Fetal Calf Serum (Hyclone Catalog # SH30071.03, lot # ATG32533)/ 1x Penicillin-streptomycin-L-Glutamine (GIbco 10378-016). Aurora black 384 well, square, clear-bottomed image quality plates (Aurora Catalog #1022-11330) were used for the assay.
On day 1:
1)Remove media with aspirator (ELX405 plate washer).
2)Add 30 microl Ham's F12/0.5% Bovine Serum Albumin (fatty acid-free) (Sigma A8806-5G) /25 mM HEPES pH 7.4 (Invitrogen) + 10 microg/mL Alexa 488-HDL with Thermo Combi fluid handler (slow speed setting)
3)Pin transfer 100 nl compounds and positive control (1 uM BLT-1).
4)Incubate 3 hours @ 37 degrees C in humidified cell culture incubator.
5)Remove media with aspirator.
6)Wash twice with 30 microl PBS (+Ca & Mg) using the Thermo Combi on slow speed setting
7)Analyze Alexa-488-HDL binding with Perkin-Elmer Envision plate reader (FITC mirror #403) with bottom read
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) and positive control wells (PC; n=20) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

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 Max_Concentration.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
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.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold 40.0
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 >= 30% but <= 70%, 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.

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.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AbsAC40_Qualifier<, =, or >String
2AbsAC40_uM*The concentration at which the fitted curve passes activity threshold 40.FloatμM
3pAbsAC40_MEqual to -1*log10(AbsAC40).Float
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0uM_(%) (0μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.000001uM_(%) (1e-06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.000002uM_(%) (2e-06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.000006uM_(%) (6e-06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.000007uM_(%) (7e-06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.000018uM_(%) (1.8e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.000021uM_(%) (2.1e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.000050uM_(%) (5e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.000060uM_(%) (6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.00016uM_(%) (0.00016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.000195uM_(%) (0.000195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.00046uM_(%) (0.00046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.00056uM_(%) (0.00056μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.00135uM_(%) (0.00135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.0016uM_(%) (0.0016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_0.0042uM_(%) (0.0042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_0.005uM_(%) (0.005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_0.012uM_(%) (0.012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_0.015uM_(%) (0.015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_0.038uM_(%) (0.038μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_0.046uM_(%) (0.046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_0.11uM_(%) (0.11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33Activity_at_0.135uM_(%) (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34Activity_at_0.35uM_(%) (0.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_0.42uM_(%) (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36Activity_at_1uM_(%) (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
37Activity_at_1.2uM_(%) (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
38Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_3.8uM_(%) (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_11uM_(%) (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_19.5uM_(%) (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_28.5uM_(%) (28.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_35uM_(%) (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_80uM_(%) (80μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 2 R01 HL052212-11

Data Table (Concise)
Classification
PageFrom: