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BioAssay: AID 1480

Profiling compound fluorescence in IgMXP3 at 488/530 nm; counter screen to single point confirmation of ABCG2 screen

For the multiplex screen of ABC transporters (AID 1325 and 1326) testing compounds with innate fluorescence would register as false postitive. Thus it was essential to assess the autofluorescence of these compounds with excitation at 488 nm and emission at 530 +/- 20 nm. These are the same conditions used for measuring the magnitude of pump substrate (flurosecent J-aggregate-forming lipophilic more ..
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 Tested Compounds
 Tested Compounds
All(273)
 
 
Active(89)
 
 
Inactive(184)
 
 
 Tested Substances
 Tested Substances
All(273)
 
 
Active(89)
 
 
Inactive(184)
 
 
 Related BioAssays
 Related BioAssays
AID: 1480
Data Source: NMMLSC (UNM_ABC_TRANSPORTERS_ABCG2_COMPOUND_FLUORESCENCE)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-01-04
Modify Date: 2011-10-07

Data Table ( Complete ):           Active    All
BioActive Compounds: 89
Depositor Specified Assays
AIDNameTypeComment
1325High-throughput multiplex screening for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screenscreeningPrimary Screen for ABCG2 in IgMXP3 cells
1326High-throughput multiplex screening for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screenscreeningPrimary Screen for ABCB1 in CCRF-Adr cells
1818Summary of High-throughput multiplex screening for ABC transporter inhibitorssummarySummary of High-throughput multiplex screening for ABC transporter inhibitors.
Description:
University of New Mexico Assay Overview:
Assay Support: 1R03MH081228-01A1
High-throughput multiplex screening for ABC transporter inhibitors
PI: Richard Larson MD, PhD
Assay Development: Irena Ivnitski-Steele PhD
Assay Implementation: Irena Ivnitski-Steele PhD, Terry Foutz, Anna Waller PhD, Mark Carter
Target Team Leader for the Center: Bruce Edwards, PhD (BEdwards@salud.unm.edu)

Assay Background and Significance:
For the multiplex screen of ABC transporters (AID 1325 and 1326) testing compounds with innate fluorescence would register as false postitive. Thus it was essential to assess the autofluorescence of these compounds with excitation at 488 nm and emission at 530 +/- 20 nm. These are the same conditions used for measuring the magnitude of pump substrate (flurosecent J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1)) present in the cell. For the flow cytometer to measure the fluorescence, the compound could be either non-specifically bound external to the cell or it could be cell permeable.
Protocol
The assay was carried out similar to AID 1325 and 1326. Micro plates were made with cells and test compounds, but without the presence of JC-1 substrate. The total volume of 15.1 microliters were dispensed sequentially as follows: 1) Assay buffer (10 microliter/well); 2) test compound (0.1 microliter/well), 3) drug-resistant unlabeled IgMXP3 cells (ABCG2)(5 microliter/well). Final in-well concentration of test compound was 6.6 micromolar and of cells was 3 million cells/ml. Assay buffer is a propriatery buffer from Cell Technology (www.celltechnology.com) for their JC-1 product (JC-1 Mitochondrial Membrane Potential Detection Kit, JC100).

The plate contents were mixed, the plate rotated end-over-end at 4 RPM and 25 degrees C for 10 minutes, and then cell samples were immediately analyzed. Sample analysis was conducted with the HyperCyt(R) high throughput flow cytometry platform [Kuckuck, et al., 2001; Ramirez, et al., 2003]. Approximately 2 microliter volumes from each well were collected at a rate of approximately 40 samples per minute. This resulted in analysis of approximately 1,000 cells of each cell type from each well. Flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) was collected via CyAn flow cytometer (Beckman Coulter). The resulting time-resolved single data file per plate was analyzed by IDLQuery software to determine the compound activity in each well.

The control wells contained DMSO instead of test compound.

Calculations:
Test compounds autofluorescence was calculated on the basis of the median fluorescence intensity (MFI) detected in the green fluorescence channel (530 +/- 20 nM) and the subtraction of the autofluorescence of the cells (IgMXP3), as shown in the following equation:
Fluorescence = MFI_CMPD - MFI_CELL
where MFI_CMPD is the MFI of cells in the presence of test compound and MFI_CELL is the MFI of cells in the presence of DMSO (control well). Two repetitions of this assessement was made, hence the mean of fluorescence is the reported value.

Compound was noted active if the fluorescence was greater than the average plus three standard deviations of the control wells on the plate. For IgMXP3 (ABCG2 expressing cells) active compounds have Fluorescence > 5.69 + 3 x 2.66, and the Activity score > 1. Activity score was calculated based on normalizing the Fluorescence to the maximum measured, i.e., Activity Score = Mean_fluorescence/Maximum.

Keywords:
NIH Roadmap, NMMLSC, flow cytometry, drug-resistance transporters, ABCB1, ABCG2

Abbreviations: nm for nanometer, RPM for revolutions per minute
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1MEAN_FLUORESCENCEMean compound fluorescence at 530 nm in IgMXP3 cells from two repetitionsFloat
2STD_FLUORESCENCEStandard deviation of compound fluorescence at 530 nm in IgMXP3 cells from two repetitionsFloat
Additional Information
Grant Number: 1R03MH081228-01A1

Data Table (Concise)
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