Profiling compound fluorescence on GSH Beads with 488 nm excitation and 530 nm emission
Innate fluorescence from testing compounds could potentially affect response measurements during screening. For the multiplex screen of BCL-2 (AID:950,951, 952, 1007, 1008, 1009) glutathione labeled beads were coated with GST Bcl-fusion proteins, onto which fluorescent Bim binds. In addition, non-protein coated glutatione beads were also present in each assayed well. The fluorescence of these more ..
BioActive Compounds: 489
University of New Mexico Assay Overview:
Assay Support: NIH 1X01 MH079850-01
HTS to identify small molecule regulators of Bcl-2 family protein interactions
PI: Larry Sklar, Ph.D.
Assay Implementatiion: Peter Simons Ph.D, Susan Young MS, Anna Waller Ph.D, Mark Carter M.S.
Target Team Leader for the Center: Larry Sklar, Ph.D., (firstname.lastname@example.org)
Assay Background and Significance:
Innate fluorescence from testing compounds could potentially affect response measurements during screening. For the multiplex screen of BCL-2 (AID:950,951, 952, 1007, 1008, 1009) glutathione labeled beads were coated with GST Bcl-fusion proteins, onto which fluorescent Bim binds. In addition, non-protein coated glutatione beads were also present in each assayed well. The fluorescence of these glutathione beads in the presence of testing compounds or just DMSO were measured with excitation at 488 nm and emission at 530 +/- 20 nm. For the flow cytometer to measure the fluorescence, the compound would be non-specifically bound externally to the bead. The fluorescence measurements were calibrated with the aid of calibration beads, which are assessed to have certain molecules of equivalent soluble fluorophore (MESF) per bead.
The multiplex assay consists of seven bead sets of glutatione labeled bead (ThermoFisher Scientific product numbers XPR-1687-XPR-1696); six of the bead sets have been pre-incubated with GST Bcl-fusion protein target and one unlabeled bead set. These bead sets are labeled with different intensities with red fluorescence, hence are distinguishable from one another. After overnight incubation with GST-tagged proteins, the beads are washed in HPSMTB buffer (30mM HEPES, 100mM KCl, 20mM NaCl, 1mM MgCl(2), 0.01% Tween-20, 0.1% BSA). The bead sets are then resuspended in HPSMTB and stored on ice until the 384-well plates are assembled using the Biomek FXp, Automated work station;(5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 100 nM F-Bim in HPSMTB). The final test compound concentration is 10 microM. The control wells contain DMSO instead of test compound.
Sample analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt(R) system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates [Kuckuck, et al. 2001]. Flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nanom (FL1) and 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Time-resolved data is acquired as a single data file that is subsequently analyzed using IDLeQuery software (software developed inhouse by Dr. Bruce Edwards) that merges flow cytometry data files with compound worklist files generated by HyperSip software. The raw data are parsed in IDLeQuery to produce annotated fluorescence summary data for each well. Gating based on forward scatter (FS) and side scatter (SS) parameters is used to identify singlet bead populations. Additional gating based on FL8 (red) emission distinguishes the beads coated with different proteins. Per bead population, the median channel fluorescence (MCF) from FL1 (green) channel is calculated.
Autofluorescence of test compounds were 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 uncoated avidin bead, as shown in the following equation:
Fluorescence = MFI_CMPD - MFI_DMSO
where MFI_CMPD is the MFI of avidin beads in the presence of test compound and MFI_DMSO is the MFI of beads in the presence of DMSO (control well).
The Fluorescence was calibrated to kMESF (kiloMESF, 1000*MESF) utilizing Bangs Labs calibration beads by the following equation:
kMESF_bead = Slope*Fluorescence + Y-intercept
where Slope and Y-intercept are from linear fit of MFI measurements of the 5 different calibration beads with known MESF.
The calculated kMESF for each compound is the reported Activity Score, with the exception of when the kMESF was greater than 100 the Score was set at 100. A compound was noted active if the fluorescence was greater than the average plus three standard deviations of the entire screen. For this assay that kMESF cutoff was 86 (25.5+3*20). Therefore any compound with Activity Score > 86 is active.
NIH Roadmap, NMMLSC, flow cytometry, compound fluorescence, Bcl-2 family
** Test Concentration.
Data Table (Concise)