For in vivo BrdU labeling, one milligram of BrdU (Sigma) in 100 μL DPBS per mouse was injected intraperitoneally, before mice were sacrificed one hour later and BrdU cooperation in various B cell subpopulations was analyzed. For in vitro labeling, BrdU was added to the cell culture at a concentration of 10 μM for 45 mins at indicated time points. BrdU staining was performed with the BrdU flow cytometry kit following manufacturer instructions (BD Biosciences). For CFSE dilution assay, the CellTrace CFSE kit (Invitrogen) was used to label cells with CFSE.
Brdu flow cytometry kit
Manufactured by BD
The BrdU flow cytometry kit is a laboratory tool used to detect and quantify cellular proliferation. It provides a method for measuring the incorporation of the synthetic thymidine analog bromodeoxyuridine (BrdU) into the DNA of dividing cells. The kit includes reagents and protocols for sample preparation, staining, and flow cytometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cm h2dcfda, by Thermo Fisher Scientific (2 mentions)
Click it edu flow cytometry assay kit, by Thermo Fisher Scientific (2 mentions)
Celltrace cfse kit, by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Annexin 5 apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by BD (1 mentions)
Click it edu flow cytometry kit, by Thermo Fisher Scientific (1 mentions)
Cd45 microbeads, by Miltenyi Biotec (1 mentions)
6 protocols using brdu flow cytometry kit
1
In Vivo and In Vitro BrdU Labeling
2
Proliferation and Apoptosis Assays
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KPf/fC or FG cells were infected with shRNA and sorted 72 h later; 50,000 transduced cells were plated in a 24-well plate in 10% DMEM. For BrdU analysis, 24 h after plating, media was refreshed with media containing BrdU (BD Biosciences); after an 18 h pulse in BrdU-containing media, cells were trypsinized, fixed, permeabilized, and stained with anti-BrdU-APC using the BrdU flow cytometry kit (BD Biosciences). For Annexin V analysis, cells were trypsinized and analyzed with the Annexin V apoptosis kit (eBioscience) 48 h after plating.
3
Analyzing Cell Cycle and Oxidative Stress
4
Analyzing Cell Cycle and Oxidative Stress
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Antibodies used in flow cytometry are mentioned in Supplementary Table 6. For cell-cycle analysis, the BrdU flow cytometry kit (BD Biosciences) or Click-iT EdU Flow Cytometry Assay Kit (Invitrogen) was used according to the manufacturer’s instructions. For evaluation of intracellular ROS levels, ALL cells were incubated for 7 min with 1 μM 5-(and 6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Invitrogen, Carlsbad, CA) at 37°C for oxidation of the dye by ROS. After washing with PBS, the cells were incubated additional 15 min at 37°C in PBS to allow complete deacetylation of the oxidized form of CM-H2DCFDA by intracellular esterases. The levels of fluorescence were then directly analyzed by flow cytometry, gated on viable cells.
5
In vitro and in vivo BrdU assays
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For in vitro bromodeoxyuridine (BrdU) assays, cells were pulsed with 1 mM BrdU for 1 hours. For in vivo BrdU assays, mice were injected with CsA (4 mg/mouse), FK506 (0.4 mg/mouse), or vehicle. At the 48 hours time-point, mice were killed and BM analyzed. BrdU (1.5 mg/mouse) was injected 1 or 18 hours before analysis. BM was lineage-depleted and progenitors populations labeled and gated as described in the sorting strategy above. Cells were fixed and labeled using a BrdU flow cytometry kit (BD Biosciences). Proliferation was also assessed by CFSE dilution (Life Technologies, Molecular Probes), cells were labeled with 2 μM CFSE following the manufacturer's protocol.
6
Longitudinal BrdU and EdU Incorporation Analysis
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For longitudinal analysis of BrdU incorporation, mice were administered BrdU in their drinking water for up to 4 wk, the maximal possible duration due to BrdU toxicity, similar to what has previously been published for LCs (Merad et al., 2002 (link)). BrdU incorporation was detected using a BrdU Flow cytometry kit (BD Biosciences) following the manufacturer’s protocol. To quantify cells currently in S-phase, EdU incorporation assays were performed. Mice were injected i.p. with 1 mg of EdU and sacrificed 14 h later. Epidermal DETCs and LCs were enriched using CD45 microbeads (Miltenyi Biotec), and EdU incorporation was detected using the Click it EdU flow cytometry kit (Life Technologies) according to the manufacturer’s instructions.
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