Alexa fluor 488 conjugated γh2ax antibody

For quantification of γH2AX, cells were washed with PBS and fixed with ice-cold 70% ethanol. After washing with PBS, cells were incubated in incubation buffer (0.5% BSA/0.25% Triton X-100 in PBS) for 15 minutes on ice and incubated with Alexa Fluor 488 conjugated γH2AX antibody (BD Biosciences: 560445) or an isotype control (BD Biosciences: 557702) according to the manufacturer’s protocol. Samples were washed with incubation buffer, resuspended in PBS, and analyzed by BD FACS Canto II using FlowJo software. A minimum of 20,000 cells within the gated region were analyzed.
For characterization of iPSCs for cell surface markers (SSEA-4, TRA-1–60, and TRA-1–81), the following antibodies were used, such as Alexa Fluor 647 Mouse anti-SSEA-4, 560796; Alexa Fluor 488 Mouse anti-Human TRA-1–60, 560173; Alexa Fluor 647 Mouse anti-Human TRA-1–81, 560124 (BD Biosciences). For detection of intracellular marker (Oct-¾), cells were fixed in 2% paraformaldehyde, permeabilized in 0.1% Triton X-100 on ice, incubated with Oct-¾ antibody (1:20; sc-5279; Santa Cruz) for 1 hour at room temperature. MSCs were characterized using Stemflow hMSC analysis kit (BD Biosciences; 562245).
For EdU flow, cells were labeled with 10 uM EdU for 30 minutes and analyzed using Click-iT assay kit (ThermoFisher Scientific). Apoptosis was analyzed using caspase-3 antibody (BD Biosciences; 559585).