Cell cycle analysis was performed using the BrdU Flow Kit according to the manufacturer protocol (BD, FITC BrdU Flow Kit; Cat. No. 559619), with cells pulsed with BrdU for 1 hour at 37°C. Annexin V apoptosis staining was performed using the Apoptosis Detection Kit according to the manufacturer protocol (BD, FITC Annexin V Apoptosis Detection Kit; Cat. No. 556547). Cells were co-stained with 4′,6-diamidino-2-phenylindole (DAPI) to measure DNA content and analyzed with a BD LSRFortessa flow cytometer (BD Biosciences) and FlowJo software (TreeStar).
Brdu flow kit
Manufactured by BD
Sourced in United States, Germany
The BrdU Flow Kit is a laboratory equipment product designed to facilitate the detection and quantification of bromodeoxyuridine (BrdU) incorporation into cellular DNA. It provides the necessary reagents and protocols for the analysis of BrdU labeling in cultured cells using flow cytometry.
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Lab products found in correlation
Bromodeoxyuridine (brdu), by BD (77 mentions)
Bromodeoxyuridine (brdu), by Merck Group (61 mentions)
Cytofix cytoperm kit, by BD (16 mentions)
Facscanto 2, by BD (15 mentions)
Lsrfortessa, by BD (12 mentions)
Facscalibur, by BD (11 mentions)
Cytofix cytoperm buffer, by BD (8 mentions)
Facscalibur flow cytometer, by BD (8 mentions)
Facscan, by BD (8 mentions)
7 aad, by BD (7 mentions)
Lsrii flow cytometer, by BD (7 mentions)
Perm wash buffer, by BD (7 mentions)
Annexin 5 apoptosis detection kit, by BD (6 mentions)
Golgistop, by BD (6 mentions)
Facsdiva, by BD (6 mentions)
Apo brdu kit, by BD (6 mentions)
Lsrfortessa flow cytometer, by BD (6 mentions)
Facsaria, by BD (5 mentions)
Cytofix cytoperm, by BD (5 mentions)
7 aminoactinomycin d, by BD (4 mentions)
453 protocols using brdu flow kit
1
Cell Cycle and Apoptosis Analysis
2
Measuring BrdU Incorporation in Microglia
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The incorporation of 5-bromo-2′-deoxyuridine (BrdU) was analyzed in mixed glial cell cultures using flow cytometry and labeling with a conjugate anti-BrdU antibody (FITC BrdU flow kit, BD Pharmingen, CA, USA). Mixed glial cell cultures were incubated with 0.25–5 μM teriflunomide and co-treated with GM-CSF (5 ng/ml; Peprotech, Hamburg, Germany) from day 5 after preparation. The next day, 10 μM BrdU (FITC BrdU flow kit, BD Pharmingen) was added for 16 h.
After microglia isolation, the Fc receptors were blocked with mouse anti-rat CD32 for 30 min on ice (clone: D34-485; BD Pharmingen). Surface staining of microglia was done with allophycocyanin (APC)-conjugated rat anti-CD11b/c antibody for 30 min at 4 °C. Cells were then fixed and permeabilized with fixation/permeabilization buffer (Foxp3 Staining Buffer Set; eBioscience) according to the manufacturer’s instructions. The samples were treated with DNase to expose incorporated BrdU (diluted to 300 μg/ml; BrdU flow kit, BD Pharmingen) for 1 h at 37 °C and finally stained with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody (30 min at RT, 1:100; BrdU flow kit, BD Pharmingen). The analysis was carried out using a FACSCalibur with CellQuest software (BD). Measurements were performed in duplicates per condition and in four independent experiments.
After microglia isolation, the Fc receptors were blocked with mouse anti-rat CD32 for 30 min on ice (clone: D34-485; BD Pharmingen). Surface staining of microglia was done with allophycocyanin (APC)-conjugated rat anti-CD11b/c antibody for 30 min at 4 °C. Cells were then fixed and permeabilized with fixation/permeabilization buffer (Foxp3 Staining Buffer Set; eBioscience) according to the manufacturer’s instructions. The samples were treated with DNase to expose incorporated BrdU (diluted to 300 μg/ml; BrdU flow kit, BD Pharmingen) for 1 h at 37 °C and finally stained with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody (30 min at RT, 1:100; BrdU flow kit, BD Pharmingen). The analysis was carried out using a FACSCalibur with CellQuest software (BD). Measurements were performed in duplicates per condition and in four independent experiments.
3
Assaying B cell development and proliferation
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For class switching assays, cell suspensions were stained with fluorochrome-conjugated anti-IgG1, anti-IgG3 (BD-Biosciences), anti-IgG2b (BioLegend), or anti-IgA (Southern Biotech). Samples were acquired on a LSRFortessa cell analyzer (BD-Biosciences). Analysis of B cell development and differentiation was performed using anti-CD21/CD35-FITC, anti-IgD-FITC, anti-IgM-PE, anti-IgM-FITC, anti-CD43-PE (BD-Biosciences) and anti-CD23-PE, anti-CD3-PE and anti-CD19-APC (BioLegend) antibodies. For cell proliferation analysis by cell tracking dye dilution, primary B cells were pulsed with 2 μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) or 5 μM CellTrace Violet (Thermofisher) for 10 min at 37C. CFSE/CellTrace covalently labels intracellular molecules, and each cell division halves the signal intensity. For cell cycle analysis, CH12 cells were collected, fixed, and permeabilized using Fixation/Permeabilization Solution (included in BrdU Flow Kit, BD-Biosciences) according to the manufacturer’s instructions. BrdU pulse and staining was performed by using BrdU Flow Kit (BD-Biosciences) according to the manufacturer’s instructions.
4
Cell Cycle and Apoptosis Analysis
5
Cell Cycle and Apoptosis Analysis
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Cell cycle analysis was performed using the BrdU Flow Kit according to the manufacturer protocol (BD, FITC BrdU Flow Kit; Cat. No. 559619), with cells pulsed with BrdU for 1 hour at 37°C. Annexin V apoptosis staining was performed using the Apoptosis Detection Kit according to the manufacturer protocol (BD, FITC Annexin V Apoptosis Detection Kit; Cat. No. 556547). Cells were co-stained with 4′,6-diamidino-2-phenylindole (DAPI) to measure DNA content and analyzed with a BD LSRFortessa flow cytometer (BD Biosciences) and FlowJo software (TreeStar).
6
Cell Cycle and Apoptosis Analysis
7
Multimodal Analysis of Apoptosis and Proliferation
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For apoptosis assay, BM cells were stained with antibodies for the surface markers, and incubated with the Annexin V-FITC antibody at room temperature for 15 minutes. DAPI (1 μg/ml) was added before analysis by FACS. For the in vitro apoptosis assay, the cells treated with the drug in vitro were directly stained with the Annexin V-FITC antibody, and DAPI was added before analysis by FACS. For caspase 3/7, 9 activity assays, HSPCs were plated into 96-well white luminescence plates and the Caspase3/7, 9 activities were analyzed according to the manufacturer’s instructions (Promega). For the mitochondrial membrane potential assay, the cells treated with the drug in vitro were determined by FACS after loading the cells with JC-1 dye according to the manufacturer’s instructions (Beyotime). For the BrdU incorporation assay, BrdU (100 mg/kg) was intraperitoneally injected, and followed by administration of BrdU (1 mg/ml) in the drinking water for 10 days. BrdU incorporation was determined by FACS analysis using the BrdU Flow Kit (BD Biosciences). For Ki67 staining, after the surface markers staining, the cells were fixed using BrdU Flow Kit and staining with Ki67 antibody (BD Pharmingen).
8
BrdU and EdU Labeling for Cell Cycle Analysis
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Mice were given a single intraperitoneal injection of 100 μL BrdU (BD Pharmingen BrdU Flow Kits, BD, 552598) 2 or 24 h before intestine isolation. BrdU staining was performed with the BrdU flow kit (BD Pharmingen). For cell cycle analysis, the epithelium was stained with EpCam-PE, BrdU-APC, and 7-AAD 2 h later after a single intraperitoneal injection of BrdU. Cell cycle was analyzed by FACS. EdU labeling was performed by following the manufacturer’s instruction (Click-iT EdU Imaging Kit, Invitrogen, C10339).
9
Multi-parameter Flow Cytometry Analysis
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Spleen, TDLN and PP single-cell suspensions were blocked with Fc receptor blocking mAb and stained with mAb including CD4, CD8, CD49b, CD3, B220, CD11c, CD80, CD86, I-A/I-E, Gr-1 (BD PharMingen, San Diego, CA) or F4/80 (AbD Serotec, Kidlington, UK) for 20 min at 4°C, then washed and analyzed using a flow cytometer (FACSAria; BD Biosciences). For the analysis of BrdU incorporation, macrophages or DCs were stimulated with YM-2A in presence of 10 μM BrdU (BrdU Flow Kit, BD PharMingen) for 24 h, and cells were stained for cell surface markers and BrdU using the BrdU Flow Kit (BD Biosciences). For intracellular staining of IFN-γ and IL-4, cells were cultured for 4 h with PMA (25 ng/ml) and ionomycin (1 μg/ml), and cytokine release was prevented by treatment with Golgi-stop (BD PharMingen). Following surface staining of CD4 or CD8, cells were fixed using the Cytofix/Cytoperm kit (BD PharMingen) and stained with mAb including IFN-γ or IL-4 (BD PharMingen).
10
In vivo T cell proliferation assay
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