Each mouse was injected intraperitoneally with 2 mg BrdU in 200 µl PBS and euthanized four hours later. Single cell suspension of thymocytes was then prepared and stained with PE-conjugated antibodies against lineage markers B220, CD4, CD8, Gr-1, Mac-1, NK1.1, and γδTCR, PECy7-conjugated anti-CD44, and APCCy7-conjugated anti-CD25. Then BrdU staining using APC-conjugated anti-BrdU antibody was performed according to the manufacturer’s instructions (BD Biosciences). Samples were examined on LSR II Flow Cytometer and data analyzed using FACSDiva software (BD Biosciences).
Apc conjugated anti brdu antibody
Manufactured by BD
Sourced in United States
The APC-conjugated anti-BrdU antibody is a fluorescent-labeled antibody used for the detection of bromodeoxyuridine (BrdU) incorporation into DNA. This antibody is conjugated to the fluorescent dye allophycocyanin (APC), which can be detected using flow cytometry or microscopy techniques. The primary function of this product is to provide a means for the identification and quantification of cells that have incorporated BrdU, a synthetic nucleoside analog, during DNA synthesis.
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Lab products found in correlation
Facsdiva software, by BD (2 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Pe conjugated anti active caspase 3 antibody, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Facscalibur analyzer, by BD (1 mentions)
5 bromo 2 deoxyuridine brdu, by BD (1 mentions)
7 aad, by BioLegend (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by BD (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Merck Group (1 mentions)
Click it edu pacific blue flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Anti notch1, by Abcam (1 mentions)
Anti hes1, by Abcam (1 mentions)
Anti lamin b, by Santa Cruz Biotechnology (1 mentions)
Anti c myc, by Santa Cruz Biotechnology (1 mentions)
Application suite imaging software, by Leica camera (1 mentions)
Gapdh blot, by Santa Cruz Biotechnology (1 mentions)
Gel logic 6000 pro imaging system, by Carestream (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
6 protocols using apc conjugated anti brdu antibody
1
Thymocyte Cell Cycle Analysis
2
Flow Cytometry Analysis of Apoptosis and Proliferation in Leukemic Cells
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Surface staining of tNGFR leukemic cells was performed with PE-conjugated anti-human CD271 antibody (BD Biosciences). Apoptosis assays were carried out as described [8 (link)], using PE-conjugated-anti-active Caspase-3 antibody (BD Biosciences). BrdU incorporation assays were performed as described [6 (link)], using APC-conjugated anti-BrdU antibody (BD Biosciences). Flow cytometry acquisitions were carried out on a FACSCalibur™ analyzer (BD Biosciences) equipped to detect 4 fluorescent parameters with the assistance of BD CellQuest Software (BD Biosciences) and data were analyzed with FlowJo Software (Tree Star). ICN1 leukemic cells were sorted on a FACSAria™III (BD Biosciences) cell sorter on the basis of tNGFR and/or GFP expression with the assistance of BD FACSDiva Software (BD Biosciences). Migration analyses were performed by videomicroscopy, as described [6 (link)].
3
BrdU Pulse Labeling and Cell Cycle Analysis
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Cells were pulse-labeled with 10 μM 5-bromo-2-deoxyuridine (BrdU, BD Pharmingen, San Diego, CA) for 90min (37°C), washed in PBS, then fixed in 70% ethanol (1-3d, −20°C). DNA was denatured (2 M HCl, 30min, room temperature) and neutralized (0.1 M Na2B4O7, 10min, room temperature). Cells were stained with APC-conjugated anti-BrdU antibody (BD Pharmingen) and 7-AAD (BioLegend, San Diego, CA), and analyzed by flow cytometry [54 (link)].
4
In vivo DNA Labeling in Mice
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For in vivo DNA labeling, mice were intraperitoneally administered 1 mg of BrdU and intravenously injected 1 mg of EdU (Sigma-Aldrich) in PBS. To detect incorporated BrdU and EdU in DNA, cells were analyzed with a BrdU assay kit for flow cytometry using an APC-conjugated anti-BrdU antibody (BD Biosciences) and a Click-iT EdU Pacific Blue flow cytometry assay kit utilizing click chemistry of azide-alkyne reactions (Thermo Fisher Scientific). The procEdUres followed the manufacturer’s protocols.
5
Neutrophil Labeling and Tracking in Mice
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BrdU labeling of endogenous neutrophils was modified from previously described (19 (link)). Briefly, neutrophil precursors in the mouse BM were labeled via intravenous (i.v.) injection of 5-bromo-2′-deoxyuridine (BrdU; 2 mg per mouse; APC (Allophycocyanin) BrdU Flow Kit; BD Biosciences. Two days after BrdU injection, mice received an i.v. injection of 100 µg CD62L antibody or PBS to block transendothelial migration and to reduce neutrophil margination. Blood samples were collected at indicated times and BM, spleen and lung samples were collected at 2 day and 4 day after BrdU injection. The samples were stained for Ly6G, Ly6C, and CD11b, followed by fixation and intracellular labelling of BrdU using an APC-conjugated anti-BrdU antibody as per manufacturer’s instructions (BD Biosciences). The number of cells were counted by a MACSQuant flow cytometer and data acquisition was done on FACSCelesta SORP flow cytometer.
6
Antibody Validation and Quantification
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The following antibodies were used for immunoanalyses: anti-RND3 (Cocalico Biologicals, Reamstown, PA, USA), anti-NOTCH1 (Abcam, Boston, MA, USA ab27526), anti-HES1 (Abcam, ab71559), anti-pHis3 (Santa Cruz, Dallas, TX, USA sc-8656), anti-c-Myc (9E10, Santa Cruz, sc-40), anti-Flag (sigma, St. Louis, MO, USA F7425), anti-Histone H3 (Rabbit, abcam, ab1791), anti-GFP (Rabbit, Santa Cruz, sc-5385), anti-LaminB (Rabbit, Santa Cruz, sc-20682), APC-conjugated anti-BrdU antibody (BD, 552598). The specificity and sensitivity of the anti-RND3 antibody was validated in our previous study 14 (link). Even protein loading for immunoblotting analysis was verified by the intensity of the GAPDH blot (Santa Cruz, sc-20357). The immunoblotting densitometry was quantified by the Gel Logic 6000 PRO Imaging System (Carestream Health, Inc. Rochester, NY, USA), and the immunofluorescent and immunohistochemical image quantifications were conducted by Leica Application Suite Imaging Software (Version 4.0, Biberach, Germany).
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