Preparation and characterization of calibration beads for sorting cells expressing a beta-lactamase gene reporter.
BACKGROUND
Modern drug discovery has been based on high-throughput screening using whole-cell assays. A prominent role has been assigned to the reporter gene technology based on a beta-lactamase and the fluorogenic substrate CCF2. Successful application of this technology requires fluorescence-activated cell sorting. We describe the preparation and characterization of calibration beads for sorting cells expressing the beta-lactamase gene using the CCF2 substrate.


METHODS
To model Forster resonance energy transfer (FRET) between the coumarin donor and the fluorescein acceptor of the CCF2 reporting dye, we used activated polystyrene beads with primary amino groups. Donor and acceptor fluorophores were attached to the beads at different ratios via succinimidyl esters. The beads were characterized with a fluorescence plate reader and a flow cytometer.


RESULTS
We prepared polystyrene beads with five different ratios of donor and acceptor fluorophores and beads that carried a donor or a receptor fluorophore alone. Fluorescence measurements demonstrated that the prepared beads well represent the FRET of CCF2 substrate.


CONCLUSIONS
We have demonstrated that the prepared beads can be successfully used for the setup of fluorescence-activated cell sorting to sort cells with CCF2 reporter substrate and the beta-lactamase reporter gene.
Cunningham
Michael E
ME
Flow Cytometry Facility and Department of Neuroscience Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Kapitskaya
Marianna
M
Petrukhin
Konstantin
K
Bednar
Bohumil
B
D000818:Animals; D016466:CHO Cells; D002138:Calibration; D002469:Cell Separation / Q000379:methods*; D006224:Cricetinae; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007769:Lactams / Q000737:chemistry*; D011120:Polyomavirus / Q000235:genetics; D011137:Polystyrenes / Q000737:chemistry; D011401:Promoter Regions, Genetic; D013050:Spectrometry, Fluorescence / Q000379:methods*; D001618:beta-Lactamases / Q000235:genetics*
C109764:CCF 2; D005452:Fluoresceins; D007769:Lactams; D011137:Polystyrenes; D049452:Green Fluorescent Proteins; D001618:beta-Lactamases