BrdU (1 mg) was injected into the peritoneal cavity, and aortas were harvested and digested 24 hours later. Cells from aortas were isolated and stained as described above, and then fixed and permeated according to the manufacturer’s protocol (BD APC-BrdU Kit, 552598). BrdU+ cells were identified by flow cytometry. In vitro labeling was done with J774A.1 cells cultured with Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Before being harvested, cells were treated with 1.0 or 3.3 μM S-HDL simvastatin for 24 hours in the presence or absence of 100 μM mevalonate (Sigma-Aldrich) and labeled with BrdU for 45 min before being harvested. Cells were stained and fixed by following the protocol provided by the manufacturer (BD APC-BrdU Kit, 552598), and detected by flow cytometry.
Apc brdu kit
Manufactured by BD
Sourced in United States
The APC BrdU kit is a laboratory equipment product that enables the detection and measurement of cell proliferation. It provides a method for labeling and identifying cells that are actively dividing.
Automatically generated - may contain errors
Lab products found in correlation
Mevalonate, by Merck Group (2 mentions)
Streptavidin apc cy7, by BD (2 mentions)
Lineage cocktail kit, by BD (2 mentions)
Cd24 apc antibodies, by BD (2 mentions)
Cd49f fitc, by BD (2 mentions)
Facscanto cytometer, by BD (2 mentions)
Cd44 pe, by BD (2 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
Anti cd16 cd32, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Novocyte, by Agilent Technologies (1 mentions)
5 bromo 2 deoxyuridine brdu, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Lsr fortessa analyser, by BD (1 mentions)
5 bromodeoxyuridine brdu, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Annexin 5 pe, by BD (1 mentions)
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Anti brdu antibody, by BioLegend (1 mentions)
17 protocols using apc brdu kit
1
BrdU Labeling of Aortic Cells
2
Quantifying Aortic Cell Proliferation
3
BrdU Incorporation Assay for Alveolar and Peritoneal Macrophages
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For in vivo analysis, BrdU was intraperitoneally administered (1 mg per 100 µl per mouse) daily for 7 days before euthanasia. BrdU incorporation in AMs and PMs was assessed using a BrdU APC kit (BD) following the manufacturer’s instructions before analysis by flow cytometry.
For in vitro analysis, BrdU (10 µM) was added to AMs on day 2 after GM-CSF (20 ng ml–1) stimulation. On day 3, AMs were fixed with 4% paraformaldehyde (PFA) and then DNA was denatured using 1.5 M HCl for 30 min at room temperature. Cells were washed with PBS then stained with anti-BrdU antibody (BioLegend). BrdU (10 µM) was added to cells at day 3 and day 5 of BMDM differentiation. BrdU incorporation was assessed by flow cytometry at day 6 using a BrdU APC kit (BD) following the manufacturer’s instructions.
For in vitro analysis, BrdU (10 µM) was added to AMs on day 2 after GM-CSF (20 ng ml–1) stimulation. On day 3, AMs were fixed with 4% paraformaldehyde (PFA) and then DNA was denatured using 1.5 M HCl for 30 min at room temperature. Cells were washed with PBS then stained with anti-BrdU antibody (BioLegend). BrdU (10 µM) was added to cells at day 3 and day 5 of BMDM differentiation. BrdU incorporation was assessed by flow cytometry at day 6 using a BrdU APC kit (BD) following the manufacturer’s instructions.
4
Microglia Proliferation and Activation Assays
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For BrdU incorporation assays, cultured microglia were resuspended in complete DMEM containing 10 ng/ml M-CSF and were cultured (0.1 × 106/well) in 24 well plates with 0.1 mM BrdU for 24 or 48 h. For CFSE dilution assays, microglia were labeled with 0.434 μM CFSE in 37°C 1 x PBS for 20 min (Thermofisher) according to the product directions and were cultured in complete DMEM containing 10 ng/ml M-CSF at 37°C for 24, 48, and 72 h. At the appropriate time point, cells were detached from plates, were washed with FACS buffer (1 × PBS with 2% FCS) and then stained and analyzed by flow cytometry as follows.
All staining steps were performed at 4°C in the dark, centrifugation steps were all at 524 × g or 5 min at 4°C, and staining volumes were 100 μl. Cells (1 × 106/stain) were first blocked with anti-CD16/CD32 (5 μg/ml) (ThermoFisher) in FACS buffer. Cells were washed in 200 μl FACS buffer and then stained for 30 min in FACS buffer containing anti-mouse CD11b (M1/70, ThermoFisher), CD45 (30-F11 ThermoFisher) and Fixable Viability Dye eFluor506 (diluted 1:1,000, ThermoFisher). Intranuclear staining for BrdU was measured using the APC BrdU kit (BD Bioscience). Cells were washed twice with FACS buffer prior to flow cytometry acquisition. Data were analyzed using Flowjo (Flowjo LLC).
All staining steps were performed at 4°C in the dark, centrifugation steps were all at 524 × g or 5 min at 4°C, and staining volumes were 100 μl. Cells (1 × 106/stain) were first blocked with anti-CD16/CD32 (5 μg/ml) (ThermoFisher) in FACS buffer. Cells were washed in 200 μl FACS buffer and then stained for 30 min in FACS buffer containing anti-mouse CD11b (M1/70, ThermoFisher), CD45 (30-F11 ThermoFisher) and Fixable Viability Dye eFluor506 (diluted 1:1,000, ThermoFisher). Intranuclear staining for BrdU was measured using the APC BrdU kit (BD Bioscience). Cells were washed twice with FACS buffer prior to flow cytometry acquisition. Data were analyzed using Flowjo (Flowjo LLC).
5
Analyzing Cell Cycle Dynamics via Flow Cytometry
6
Analyzing Cell Cycle Dynamics via Flow Cytometry
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All flow cytometry was performed on a FACSCanto cytometer (BD Biosciences). Mouse cell surface marker staining was determined with the Lineage Cocktail kit and streptavidin-APC-Cy7 in combination with CD44-PE, CD49f-FITC, and CD24-APC antibodies (BD Biosciences). For co-culturing experiments, 2×105 cells of each genotype were plated in 60 mm dishes, grown overnight, then treated with 100 μM BrdU for 35 minutes before staining with the APC BrdU kit (BD Biosciences). BrdU incorporation was measured in GFP− and GFP+ cells.
7
Measurement of Proliferation in ASCs
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Pro, Fasted, Refed and D0 ASCs were incubated in their respective medium for 8 h before addition of BrdU (10 µM final concentration) and further incubation for 18 h. Controls without BrdU were performed for each condition. Cells were then processed for BrdU and DNA labeling using an APC BrdU kit (BD pharmingen, BDB552598) following manufacturer’s instructions. The flow cytometry analyses were performed on a NovoCyte (Acea Biosciences Inc.). All gatings are presented in Supplementary Fig. 11 .
8
Cell Cycle and Apoptosis Analysis
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Cell cycle was analyzed with the APC BrdU Kit (BD Pharmingen) according to manufacturer’s instructions. Cells were pulsed with 10mM with bromodeoxyuridine (BrdU) for 4 h at 37°C. Cells were washed in the staining buffer, fixed/permeabilized with the Cytofix/Cytoperm buffer or Cytofix/Cytoperm buffer plus and washed with the Perm/Wash buffer. After permeabilization, cells were treated with DNase for 1 h at 37°C, and then stained with allophycocyanin (APC)- conjugated anti-BrdU antibody and 7-amino-actinomycin D (7-AAD; 25 mg/mL).
Cell apoptosis was measured using APC-Annexin V and propidium iodide (PI) staining kit following the manufacture’s protocols. Briefly, cells with different treatments were collected and washed twice with cold PBS, and then re-suspended in 1×binding buffer from the kit. APC-Annexin V and PI were added and incubated for 15 min at room temperature in the dark.
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9
Cell Cycle Analysis with APC BrdU Kit
10
BrdU Proliferation Assessment in Mice
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For assessment of TEFF proliferation by BrdU, mice were given 1 mg of BrdU intraperitoneally at day 5 and 8 p.i. and sacrificed 12–16 h hr later. BrdU staining was carried out with the APC BrdU Kit (BD Biosciences) according to the manufacturer’s instructions.
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