Cells were synchronized by incubation (48 hr) in serum-free DMEM-H21 media, released into 10% serum-containing media, collected at the indicated time points, fixed and stained with phospho (Ser 28) histone H3.3 antibody (Cell Signaling), and analyzed by FACS. For BrdU incorporation analysis, released cells were incubated with BrdU (10 μM, 30 min), fixed, and stained in PBS containing 20 μg/ml anti-BrdU (Roche). Cell cycle distribution was assessed in fixed, propidium iodide-labeled cells by FACS in combination with Flowjo software (Treestar). DNA content was measured by DAPI staining and quantitative FACS analysis. Bright field microscopy was performed to monitor the cell size. Centrosome number was assessed using a pericentrin-specific antibody (Abcam) followed by a fluorescent secondary antibody (anti rabbit Alexa 488, Invitogen) and 40 × immunofluorescence microscopic analysis of >400 cells per group.
Phospho ser 28 histone h3.3 antibody
Manufactured by Cell Signaling Technology
Phospho (Ser 28) histone H3.3 antibody is a lab equipment product that specifically recognizes and binds to histone H3.3 when phosphorylated at serine 28. This antibody can be used to detect and quantify this post-translational modification in various cellular and experimental applications.
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Lab products found in correlation
2 protocols using phospho ser 28 histone h3.3 antibody
1
Cell Cycle Progression Analysis
2
Synchronized Cell Cycle Analysis
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Cells were synchronized by incubation for 48 hrs in serum-free DMEM-H21 media followed by a release in 10% FBS-containing media and collection at the indicated time points. In some experiments, cells were also incubated with DASA-58 (40 μM, 3 hrs, Millipore) prior to harvest. Mitotic cells were collected from the media after a 2-3 min shake of the flasks 24 hrs after serum-stimulation, with a greater than 90% purity of the population. For mitotic count, cells were fixed and stained with phospho (Ser 28) histone H3.3 antibody (Cell Signaling) and analyzed by flow cytometry. Cell cycle distribution was assessed in fixed, propidium iodide-labeled cells subjected to flow cytometry using a FACSCalibur (BD Biosciences) in combination with Flowjo software (Treestar) (15 (link)).
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