For flow cytometry analyses a minimum of 105 cells from each condition was washed with PBS and then fixed in pre-cooled 70% absolute ethanol and incubated at −20°C for 60 min. Fixed cells were washed 3 times with cold PBS and then stained with Alexa Fluor 647 anti-H2A.X-phosphorylated (Ser139) antibody (no. 613408; BioLegend, Inc., San Diego, CA, USA). Finally, the cells were washed once with PBS and resuspended in 50 μl of propidium iodide (PI)/RNase staining solution (no. 4087; Cell Signaling Technology, Inc.) 15 min before fluorescence acquisition with BD FACS Canto II cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
Alexa fluor 647 anti h2a x phosphorylated ser139 antibody
Manufactured by BioLegend
Sourced in United States
Alexa Fluor 647 anti-H2A.X-phosphorylated (Ser139) antibody is a fluorescently labeled antibody that specifically binds to the phosphorylated form of the histone H2A.X protein at serine 139. This antibody can be used to detect and quantify DNA double-strand breaks in cells.
Automatically generated - may contain errors
2 protocols using alexa fluor 647 anti h2a x phosphorylated ser139 antibody
1
Phospho-H2A.X Flow Cytometry Protocol
2
Cytometric Analysis of DNA Damage
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For the cytometric analysis of H2AX phosphorylation (on serine 139), cells exposed to 2 cycles (48 h) of hyperoxia, and/or radiation and harvested in DMEM, and washed in cold PBS twice, at 1500 rpm, at 4 °C, for 5 min. Cell pellet was resuspended in ice cold solution of 70% ethanol and the samples were incubated at −20 °C for 1 h. Cells were washed twice in cold FACS buffer (10% FBS, 0.1% NaN3 in PBS) and collected by centrifugation. H2AX phosphorylation was evaluated by incubating C10 cells with Alexa Fluor® 647 anti-H2A.X-Phosphorylated (Ser139) Antibody (Biolegend, San Diego, CA, USA), overnight at 4 °C following the manufacturer’s instructions for the appropriate dilution, in FACS buffer. Next day, cells were washed three times in cold FACS buffer and the cells were resuspended at 0.5 × 107–1 × 107 cells/mL of FACS buffer. Samples were kept on ice and protected from light until analysis in a FACSCanto system (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo V10.1 software.
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