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Anti brdu fitc antibody

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The Anti-BrdU-FITC antibody is a fluorescently labeled antibody that binds to bromodeoxyuridine (BrdU), a synthetic nucleoside that can be incorporated into DNA during cell division. This antibody is commonly used in flow cytometry and microscopy applications to detect and quantify cell proliferation.

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Lab products found in correlation

Bromodeoxyuridine (brdu), by Merck Group (2 mentions) Facscanto 2 flow cytometer, by BD (2 mentions) Axioplan 2 fluorescent microscope, by Zeiss (2 mentions) Propidium iodide, by BD (1 mentions) Facscanto, by BD (1 mentions) Facsdiva, by BD (1 mentions) Propidium iodide, by Merck Group (1 mentions)

Spelling variants of this product

Anti-BrdU antibody (13 citations) FITC-conjugated anti-BrdU antibody (8 citations) Anti-BrdU-FITC (7 citations) Anti-BrdU (6 citations)

5 protocols using anti brdu fitc antibody

1

Cell Cycle and Apoptosis Analysis by Flow Cytometry

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For BrdU-PI flow cytometry, cells were labeled with 10 μM BrdU (Sigma-Aldrich) for 1 h. Cells were collected, washed with ice-cold PBS and fixed in 80% ethanol overnight at -20 °C. Cells were washed once with cold PBS and resuspended in 2 M HCl, 0.5% Triton X-100 for 30 min at room temperature. The cell suspension was neutralized in 0.1 M Na2B4O7 (pH 8.5), and cell pellets were resuspended in 100 μl 1% BSA in PBS-T (0.5% Tween-20 in PBS) containing anti-BrdU-FITC antibody (BioLegend) for 30 min at room temperature in the dark. Cells were washed once with 1% BSA in PBS-T. Cell pellets were resuspended in PBS with RNase A (24 μg/ml) and propidium iodide (54 μM), and incubated for 30 min at 37 °C.
For annexin-PI flow cytometry, the medium in which the cells were cultured was combined with the cells. The cells were washed once with ice-cold PBS, resuspended in 100 µl Annexin V Binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) with 2 µl Annexin V/Pacific Blue dye, and incubated for 15 min in the dark at room temperature. 400 µl Annexin V Binding buffer with propidium iodide (18.5 µM) was added. Samples were stored cold in the dark until analysis.
Flow cytometry was done on a BD FACSCanto II flow cytometer. Data were analyzed using BD FACSDIVA software and FlowJo (version 8.8.6).
Adhikari B., Bozilovic J., Diebold M., Schwarz J.D., Hofstetter J., Schröder M., Wanior M., Narain A., Vogt M., Stankovic N.D., Baluapuri A., Schönemann L., Eing L., Bhandare P., Kuster B., Schlosser A., Heinzlmeir S., Sotriffer C., Knapp S, & Wolf E. (2020). PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase. Nature chemical biology, 16(11), 1179-1188.
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2

Cell Cycle Analysis of DAC-Treated HCT-116 Cells

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HCT-116 cells were grown into complete medium (DMEM) and treated with DAC up to 48 hours. The DNA content was determined staining cells with 4μg/ml of propidium iodide (PI, SIGMA-Aldrich, Italy). The PI solution was prepared in PBS and it was supplemented with 40μg/ml RNase. Analysis of PI labelled cells was conducted as described previously [14 (link)] and samples were analysed on a FACSCanto (Becton Dickinson).
Bivariate analysis to evaluate cell cycle progression/duration where done by pulse labelling (1 h) cells with bromodeoxyuridine (BrdU 10μM). Analysis of BrdU labelled cells was conducted as described previously [14 (link)] [35 (link)]; briefly cells were fixed and stained with anti-BrdUFITC antibody (biolegend USA), to detect BrdU positive cells (S-phase) and propidium iodide (PI) to assess DNA content and samples were analyzed on a FACSCanto (Becton Dickinson). Experiments were repeated at least twice and 10,000 events were analysed by using the softwares: FACSDiva (Becton Dickinson) and ModFit (Verity Software House, Inc.).
Costa G., Barra V., Lentini L., Cilluffo D, & Di Leonardo A. (2016). DNA demethylation caused by 5-Aza-2′-deoxycytidine induces mitotic alterations and aneuploidy. Oncotarget, 7(4), 3726-3739.
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3

BrdU Incorporation Assay for Cell Proliferation

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For the estimation of cell proliferation, cells were allowed to attach on glass coverslips before labeling with 50 μM BrdU for 48 h, as reported before [19 (link),41 (link),42 (link)]. Cells were then fixed with 4% (v/v) formaldehyde in PBS for 10 min, permeabilized with 0.2% (v/v) Triton X-100 in PBS for 10 min and denaturation of DNA was achieved after treatment with 2 N HCl for 30 min. After blocking with 0.5% (v/v) gelatin in PBS, samples were incubated with an anti-BrdU-FITC antibody purchased from BioLegend (SanDiego, CA, USA) at 4 °C and counterstained with 2 μg/mL DAPI in PBS for 20 min. The percentage of cells with a proliferative potential in a given cell population was calculated by dividing the number of BrdU-positive nuclei by the number of the DAPI-positive nuclei, as counted under a Zeiss Axioplan 2 fluorescent microscope.
Mavrogonatou E., Papadopoulou A., Fotopoulou A., Tsimelis S., Bassiony H., Yiacoumettis A.M., Panagiotou P.N., Pratsinis H, & Kletsas D. (2021). Down-Regulation of the Proteoglycan Decorin Fills in the Tumor-Promoting Phenotype of Ionizing Radiation-Induced Senescent Human Breast Stromal Fibroblasts. Cancers, 13(8), 1987.
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4

BrdU Proliferation Assay Protocol

Cited 1 time
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The proliferative potential of the cells was estimated after labeling with 5-bromo-2’-deoxyuridine (BrdU), as previously described [27 (link)]. Briefly, cells were plated sparsely on sterile glass coverslips in DMEM containing 10% (v/v) FBS. BrdU (50 μM) was added to the cell culture medium for a period of 48 h. Cells were fixed with freshly prepared 4% (v/v) formaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized with 0.2% (v/v) Triton X-100 in PBS for 10 min, treated with 2 N HCl for 30 min and incubated with an anti-BrdU-FITC antibody from BioLegend (SanDiego, CA, USA) overnight at 4 °C. Cells were then counter-stained with 2.0 μg/mL 4’,6-diamino-2-phenylindole (DAPI) dihydrochloride in PBS for 10 min. Labeled nuclei were observed under a Zeiss Axioplan 2 fluorescent microscope (Zeiss, Jena, Germany).
Kwiatkowska K.M., Mavrogonatou E., Papadopoulou A., Sala C., Calzari L., Gentilini D., Bacalini M.G., Dall’Olio D., Castellani G., Ravaioli F., Franceschi C., Garagnani P., Pirazzini C, & Kletsas D. (2023). Heterogeneity of Cellular Senescence: Cell Type-Specific and Senescence Stimulus-Dependent Epigenetic Alterations. Cells, 12(6), 927.
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5

Cell Cycle and Apoptosis Analysis by Flow Cytometry

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For BrdU-PI flow cytometry, cells were labeled with 10 μM BrdU (Sigma-Aldrich) for 1 h. Cells were collected, washed with ice-cold PBS and fixed in 80% ethanol overnight at -20 °C. Cells were washed once with cold PBS and resuspended in 2 M HCl, 0.5% Triton X-100 for 30 min at room temperature. The cell suspension was neutralized in 0.1 M Na2B4O7 (pH 8.5), and cell pellets were resuspended in 100 μl 1% BSA in PBS-T (0.5% Tween-20 in PBS) containing anti-BrdU-FITC antibody (BioLegend) for 30 min at room temperature in the dark. Cells were washed once with 1% BSA in PBS-T. Cell pellets were resuspended in PBS with RNase A (24 μg/ml) and propidium iodide (54 μM), and incubated for 30 min at 37 °C.
For annexin-PI flow cytometry, the medium in which the cells were cultured was combined with the cells. The cells were washed once with ice-cold PBS, resuspended in 100 µl Annexin V Binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) with 2 µl Annexin V/Pacific Blue dye, and incubated for 15 min in the dark at room temperature. 400 µl Annexin V Binding buffer with propidium iodide (18.5 µM) was added. Samples were stored cold in the dark until analysis.
Flow cytometry was done on a BD FACSCanto II flow cytometer. Data were analyzed using BD FACSDIVA software and FlowJo (version 8.8.6).
Adhikari B., Bozilovic J., Diebold M., Schwarz J.D., Hofstetter J., Schröder M., Wanior M., Narain A., Vogt M., Stankovic N.D., Baluapuri A., Schönemann L., Eing L., Bhandare P., Kuster B., Schlosser A., Heinzlmeir S., Sotriffer C., Knapp S, & Wolf E. (2020). PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase. Nature chemical biology, 16(11), 1179-1188.
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