Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Genome-edited cell lines for high-throughput screening

doi: 10.1007/978-1-4939-7724-6_1

Figure Lengend Snippet: Reporter Cassette Configurations for genome editing insertion at 3’ UTR

Article Snippet: 4 , Reagent , 4 μL , Add One-Glo EX FLuc luminescence reagent (Promega) with BioRaptr FRD.

Techniques: Luciferase, FACS, Selection

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Genome-edited cell lines for high-throughput screening

doi: 10.1007/978-1-4939-7724-6_1

Figure Lengend Snippet: 1536-well plate layout for cell density and control compound optimization. Cells plated at four different cell densities across quadrants of a 1536-well white solid-bottom, plate. Control compounds at high concentration and 16-pt titrations prepared and added to cells with pintool transfer of 23 nl per well. DMSO vehicle control, PTC124 used as FLuc reporter control, Cilnidipine used as NLuc reporter control, Bortezomib used as biological pathway control, and Digitonin used as cytotoxicity control. (Right side) FLuc luminescence plate image of cell density and control compound optimization from ViewLux CCD-based plate reader. Increased luminescence is observed as cell density increases. At low cell densities increased noise, well to well variability, is observed. In loss of signal assays, decreased luminescence (darker wells) is observed in columns treated with respective controls. Titration responses are observed with various potencies between control compounds but consistent titrations across cell densities. Titration of PTC124 in columns 15, 27, and 39 demonstrate the expected inhibition of FLuc enzyme at high concentrations of the compound followed by enzyme stabilization at moderate concentrations indicating appropriate integration of the reporter gene in the cells. Viewlux settings: 30 sec exposure, high gain, slow speed, 2x binning. Image is auto-scaled based on highest and lowest signal on the plate.

Article Snippet: 4 , Reagent , 4 μL , Add One-Glo EX FLuc luminescence reagent (Promega) with BioRaptr FRD.

Techniques: Control, Concentration Assay, Titration, Inhibition

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Genome-edited cell lines for high-throughput screening

doi: 10.1007/978-1-4939-7724-6_1

Figure Lengend Snippet: Protocol table for miniaturization of genome-edited coincidence reporter cell lines and control compound optimization to 1536-well qHTS format.

Article Snippet: 4 , Reagent , 4 μL , Add One-Glo EX FLuc luminescence reagent (Promega) with BioRaptr FRD.

Techniques: Control, Concentration Assay, Titration, Incubation, Lysis

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Genome-edited cell lines for high-throughput screening

doi: 10.1007/978-1-4939-7724-6_1

Figure Lengend Snippet: Concentration response curves (CRCs) for the four control compounds tested across different cell densities for a given genome-edited FLuc-P2A-secNLuc cell line. FLuc CRCs are shown with gray symbols and NLuc CRCs are shown with blue symbols. Data was plotted and curves fit in Prism software (GraphPad). FLuc specific stabilization with PTC124 was observed as bell-shaped curves at the three highest cell densities, while NLuc stabilization was only observed with Cilnidipine at the highest cell density. Both Bortezomib, the biological control in this assay, and Digitonin, the cytotoxicity control, demonstrated consistent concordant IC50 values at the three highest cell densities. Based on these results 1500 cells/well would be selected as the best cell density with which to use in future qHTS. Error bars represent the standard deviation of two technical replicates.

Article Snippet: 4 , Reagent , 4 μL , Add One-Glo EX FLuc luminescence reagent (Promega) with BioRaptr FRD.

Techniques: Concentration Assay, Control, Software, Standard Deviation

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Genome-edited cell lines for high-throughput screening

doi: 10.1007/978-1-4939-7724-6_1

Figure Lengend Snippet: Representative statistical summary of genome-edited FLuc-P2A-secNLuc coincidence reporter cell line tested at four cell densities, each optimized with control compounds in 1536-well qHTS format and presented for the individual reporters. Averages and standard deviations were calculated from 32 (DMSO, PTC124, Cilnidipine, Bortezomib, and Digitonin treated) or 96 (untreated) samples per cell density.

Article Snippet: 4 , Reagent , 4 μL , Add One-Glo EX FLuc luminescence reagent (Promega) with BioRaptr FRD.

Techniques: Control