Click it plus edu alexa fluor 647 assay kits

For competition cell culture assays, Cas9- or dCas9-Krab–expressing cells were transduced with the ipUSEPR sgRNA (RFP-positive) constructs in 96-well plates at ~50% infection. The cell viability and the percentage of RFP-positive were obtained by high-throughput flow cytometry and 4′,6-diamidino-2-phenylindole (Invitrogen) dye exclusion. Cell cycle was monitored by Click-iT Plus EdU Alexa Fluor 647 assay kits (C10634, Invitrogen). Data were obtained by high-throughput flow cytometry using an Attune NxT flow cytometer with an autosampler (Thermo Fisher Scientific).

Flow cytometric data were collected on an Attune NxT flow cytometer with an autosampler (ThermoFisher Scientific). For the RFP-coupled growth competition assay, the ipUSEPR vector system, which expresses an sgRNA together with a TagRFP fluorescent protein, was used to infect the Cas9⁺ cells at a ~50% transduction rate. The percentage of cells with an RFP fluorescence signal (RFP⁺%) was normalized to the RFP⁺% on day 0 (48 h after lentiviral infection). The cell cycle was monitored by EdU incorporation (10 µM EdU at 37 °C for 2 h) using Click-iT Plus EdU Alexa Fluor 647 Assay Kits (C10634, Invitrogen). Apoptotic cells were detected based on the Annexin V⁺/DAPI⁻ population using an Annexin V Apoptosis Detection Kit (50-112-9048, Invitrogen). Live cells were defined by exclusion of 4′,6-diamidino-2-phenylindole (DAPI; D1306, Invitrogen) DNA staining. Cell surface integrin αVβ5 was recognized by a mouse monoclonal anti-human αVβ5 antibody (clone P1F76; sc-13588, Santa Cruz Biotech; 1:200) and stained with AF488-conjugated donkey anti-mouse IgG (ab150105, Abcam) secondary antibody. Another mouse monoclonal anti-human αVβ5 antibody (clone P1F6; 920005, BioLegend; AF647-conjugated) was used to validate the αVβ5 flow cytometry results.