APC BrdU Flow Kit

Protocol tips

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	Incubate cells with antibodies for 15 minutes on ice.

Protocol tips
Incubate cells with antibodies for 15 minutes on ice.

Publication protocol

BrdU incorporation and DNA content were analyzed with the APC BrdU flow kit (BD Pharmingen). Annexin-V–PE (BD Pharmingen) and 7-aminoactinomycin D (7-AAD) (BioLegend) stainings were performed as recommended. Cells were analyzed on a FACSCalibur flow cytometer using the CellQuest software (BD Pharmingen). FlowJo 9.5.2 software (TriStar) was used for data analysis and plot rendering. Clonogenicity assays in soft agar were performed as previously described (22). Agar plates were incubated at 37 °C for 7–14 d. GFP+ colonies were counted using a fluorescence microscope (Nikon Eclipse TS100). 3′-UTR–reporter assays were performed in HEK293T cells upon transient transfections by calcium phosphate precipitation method (40) or by polyethylenimine (Polysciences) (41). Firefly and Renilla luciferase activities were measured 48 h after transfection using the Dual-Luciferase Reporter Assay Kit (Promega), according to the manufacturer’s instructions.

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Manufacturer protocol

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