Preparation and characterization of calibration beads for sorting cells expressing a β‐lactamase gene reporter

Abstract

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 b-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 b-lactamase gene using the CCF2 substrate. Methods: To model F€ orster 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.

Channel Interactions and Robust Inference for Ratiometric β-Lactamase Assay Data: A Tox21 Library Analysis

“…Overall, only 50.7% (3482/6866) of background repressors are considered cytotoxic based on the Cell Titer Glo viability screen. While, multiple cytotoxicity and cell viability screens can be used together to capture different pathways and markers of cytotoxicity, a review of BLA assay-development literature suggested that BLA activity may decrease background fluorescence by converting green cells to blue without affecting the cell number. − As BLA breaks down CCF2/4, coumarin concentration increases, and the cell changes color from green to blue. , The change in color corresponds to increase in 460 nm fluorescence and may lead to decreased background fluorescence because fewer cells are now green, while the total number of cells remains constant. , Thus, strong BLA upregulation is expected to turn many cells blue and may decrease fluorescence at 530 nm without cytotoxicity. Indeed, 67.6% of chemicals that induce channel 2 response with E MAX above 50% of positive control activity and AC 50 below −4.5 log 10 M units repress background activity.…”

“…40−42 As BLA breaks down CCF2/4, coumarin concentration increases, and the cell changes color from green to blue. 40,41 The change in color corresponds to increase in 460 nm fluorescence and may lead to decreased background fluorescence because fewer cells are now green, while the total number of cells remains constant. 40,41 Thus, strong BLA upregulation is expected to turn many cells blue and may decrease fluorescence at 530 nm without cytotoxicity.…”

“…40,41 The change in color corresponds to increase in 460 nm fluorescence and may lead to decreased background fluorescence because fewer cells are now green, while the total number of cells remains constant. 40,41 Thus, strong BLA upregulation is expected to turn many cells blue and may decrease fluorescence at 530 nm without cytotoxicity. Indeed, 67.6% of chemicals that induce channel 2 response with E MAX above 50% of positive control activity and AC 50 below −4.5 log 10 M units repress background activity.…”

“…The well-to-well differences in cell and dye loading are expected to be random, while concentration-dependent channel 1 trends are often interpreted as cytotoxic effects. , However, previous analysis of Tox21 BLA assays and our analysis of channel 1 and Cell Titer Glo viability counterscreen shows that in the case of Tox21 data loss of channel 1 fluorescence did not correlate well with loss of cell viability as indicated by the ATP concentration. Furthermore, the literature review suggested that the channel 1 fluorescence may decrease due to BLA activity and without cytotoxic effects. − …”

Recent Developments of Biological Reporter Technology for Detecting Gene Expression

“…The assay could be used to specifically image endogenous cell-surface furin, urokinase plasminogen activator metallo-protease activity. Cunnningham et al explained that polystyrene beads could be successfully utilized for establishing fluorescent-activated cell sorting to sort cells with the Bla reporter gene by using the substrate CCF2 (Cunningham et al, 2005). Lippard demonstrated the efficient screening over 3600 reaction products of platinum based antitumor drugs for their ability to inhibit transcription of Bla in the BlaM HeLa cell line by monitoring the cleavage of CCF2/AM.…”