Profiling compound fluorescence in CCRF-Adr at 488/530 nm; counter screen to single point confirmation of ABCB1 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 ..
BioActive Compounds: 83
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.
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 CCRF-Adr cells (ABCB1)(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.
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 (CCRF-Adr), 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 CCRF-Adr (ABCB1 expressing cells) active compounds have Fluorescence > 10.34 + 3 x 4.84, and the Activity score > 2. Activity score was calculated based on normalizing the Fluorescence to the maximum measured, i.e., Activity Score = Mean_fluorescence/Maximum.
NIH Roadmap, NMMLSC, flow cytometry, drug-resistance transporters, ABCB1, ABCG2
Abbreviations: nm for nanometer, RPM revolutions per minute
** Test Concentration.
Data Table (Concise)