BM cells were incubated with antibodies against cell surface markers (panel P16, table S7) and were additionally stained for BrdU using the Fluorescein Isothiocyanate BrdU Flow Kit from BD Biosciences (559619; RRID: AB_2617060) according to the manufacturer’s instructions. Briefly, cells were washed in PBS + 2% FBS, fixed/permeabilized in cytofix/cytoperm solution, washed with Perm/Wash, and incubated with the anti-BrdU antibody overnight at 4°C. DNase treatment was used to expose the BrdU epitope. To determine the DNA content, BrdU-stained cells were incubated with DAPI.
Fluorescein isothiocyanate brdu flow kit
Manufactured by BD
The Fluorescein Isothiocyanate BrdU Flow Kit is a laboratory reagent used for the detection and analysis of DNA synthesis and cell proliferation in flow cytometry applications. The kit contains fluorescein isothiocyanate (FITC) labeled anti-BrdU antibody and 7-AAD for cell cycle analysis.
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2 protocols using fluorescein isothiocyanate brdu flow kit
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BrdU Incorporation Assay for Cell Proliferation
2
BrdU Incorporation Assay for Cell Cycle Analysis
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For the BrdU incorporation assay, control or siRNA-treated cells were incubated with BrdU for 1 hour and the assay was performed using the fluorescein isothiocyanate BrdU Flow Kit (cat 559619; BD Biosciences, San Jose, CA), following the manufacturer’s instructions. Briefly, the cells were fixed after removing the medium with BrdU. Then, DNase was used to expose incorporated BrdU. Next, the anti-BrdU antibody was added and bound to newly synthesized cellular DNA, which is labeled with BrdU. 7-aminoactinomycin D was used for total DNA staining. Measurement of cell-cycle parameters was performed with the Becton Dickinson LSRII Flow Cytometer (BD Biosciences, San Jose, CA) and acquired data were analyzed by BD FACSDiva software 8.0 and FlowJo V10 (BD Biosciences). Each experiment was conducted 3 times.
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