Anti brdu

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-BrdU is a monoclonal antibody specifically designed to detect the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into cellular DNA. BrdU is a synthetic nucleoside analog that is commonly used to label proliferating cells in DNA synthesis studies. The Anti-BrdU antibody binds to the BrdU incorporated into the DNA, allowing for the identification and quantification of cells undergoing DNA replication.

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20 protocols using anti brdu

Multiparametric Flow Cytometry Immunophenotyping

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Cells were stained with anti-BrdU (PRB-1), anti-CD4 (RM4-5), anti-CD11c, anti-CD11b (M1/70), anti-CD25 (PC61), anti-Ly6G (Gr1), anti-CD44 (IM7), anti-CD45.1 (A20), anti-Thy1.1 (HIS51), anti-Thy1.2 (30-H12), anti-IL-17A (eBio17B7), anti-IFN-γ (XMG1.2), anti-MHCII (M5/114.15.2) (all Abs from eBioscience) and anti-GM-CSF, (140706, BD PharMingen, San Diego, CA). Cells were acquired using a FACS LSR II (BD Biosciences) and analyzed using a FlowJo software (Treestar, Ashland, OR).

Do J.S., Asosingh K., Baldwin WM I.I.I, & Min B. (2014). IFNγR signaling in non-T cell targets regulates T cell-mediated intestinal inflammation through multiple mechanisms. Journal of immunology (Baltimore, Md. : 1950), 192(6), 2537-2541.

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Multiparameter Flow Cytometry Assay for Immune Cell Profiling

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We used the following antibodies and reagents: anti-CD4 (clone: GK1.5)-FITC, -PerCP-Cy5, or -APC, anti-CD8 (clone: 53-6.7)-FITC, -PerCP-Cy5, or -APC, anti-IFN-γ (clone: XMG1.2)-APC, anti-CD44 (clone: IM7)-APC, anti-BrdU (clone: Bu20a)-PE, 7-AAD, anti-IL12 (clone: C15.6)-PE, anti-IL4 (clone: 11B11)-PE, anti-IL22 (clone: Poly5164)-PE, anti-IL10 (clone: JES5-16E3)-PE, anti-IL9 (clone: MH9A4)-PE, and anti-CD69 (clone: H1.2F3)-PE (all from eBioscience); anti-caspase-3 (clone: C92605)-FITC (From BD Biosciences); anti-FasL (SC6237) (Santa Cruz Biotechnology); and IgG-Fab2 (clone: 4412) (Cell Signaling).

Tousif S., Singh D.K., Ahmad S., Moodley P., Bhattacharyya M., Van Kaer L, & Das G. (2014). Isoniazid Induces Apoptosis Of Activated CD4+ T Cells: IMPLICATIONS FOR POST-THERAPY TUBERCULOSIS REACTIVATION AND REINFECTION. The Journal of Biological Chemistry, 289(44), 30190-30195.

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BrdU Incorporation and Flow Cytometry

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All fixation and permeabilization reagents were obtained from BD Biosciences. Mice were injected intraperitoneally with 2 mg of BrdU (Sigma, St Louis, MO) dissolved in saline. 15 hrs later, spleens were harvested and single cell suspensions prepared. Cells were incubated with antibodies specific for cell surface markers, washed and resuspended in Cytofix/Cytoperm. After 20 min at RT, cells were washed with Perm/Wash, incubated in Cytoperm Plus for 10 min at RT, washed again with Perm/Wash and resuspended in Cytofix/Cytoperm. After 5 min at RT, cells were washed with Perm/Wash, resuspended in PBS containing 300 ug/ml of DNase I (Sigma) and incubated for 1.5 hrs at 37°C in a 5% CO₂ incubator. After washing with Perm/Wash, cells were stained with anti-BrdU (eBioscience; clone# BU20A) and processed for flow cytometric analysis.

Gorman J.V., Starbeck-Miller G., Pham N.L., Traver G.L., Rothman P.B., Harty J.T, & Colgan J.D. (2014). Tim-3 directly enhances CD8 T cell responses to acute Listeria monocytogenes infection. Journal of immunology (Baltimore, Md. : 1950), 192(7), 3133-3142.

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Quantifying Infarction Volumes and Remodeling

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Infarction volumes were quantified on Nissl-stained sections using the “indirect” morphometric method. Immunohistochemistry was performed as described before^{6 (link)}. To assess microvessel remodeling, double-labeling of anti-type IV Collagen (1:10, SouthernBiotech) with anti-Ki67 (1:500, Abcam) (a general cell proliferation marker) was pursued as a surrogate marker of angiogenic-related events. To study neurogenic-related events, we double-stained anti-DCX (1:100, Abcam) with anti BrdU (1:50, Invitrogen) as a surrogate marker of neurogenesis.
To clarify that Ki67 positive cells are not proliferating microglia/macrophages we double-stained Ki67and Iba1.

Esposito E., Hayakawa K., Maki T., Arai K, & Lo E.H. (2015). Effects of postconditioning on neurogenesis and angiogenesis during the recovery phase after focal cerebral ischemia. Stroke; a journal of cerebral circulation, 46(9), 2691-2694.

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Immunofluorescence Staining of Cells

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Cells were fixed in 4% paraformaldehyde first, then permeabilized with 0.1% Triton X-100 and blocked with 5% bovine serum albumin (Solarbio, China). Anti-Flag (1:1000; Origene, USA), anti-SATB2 (1:10; Abcam, USA) and anti-BrdU (1:1000; Invitrogen, USA) were used for overnight incubation at 4℃, and fluorescent secondary antibody (red) was used for 1 h incubation at room temperature. At last, the slides were mounted with mounting media containing DAPI (blue) to stain the nuclei before observed with a laser scanning confocal microscope (Zeiss, Germany). For BrdU assay, three independent samples were used for each group. Cell numbers of BrdU-positive cells and total cells were counted in 5 images per sample.

Xin T., Li Q., Bai R., Zhang T., Zhou Y., Zhang Y., Han B, & Yang R. (2021). A novel mutation of SATB2 inhibits odontogenesis of human dental pulp stem cells through Wnt/β-catenin signaling pathway. Stem Cell Research & Therapy, 12, 595.

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BrdU Incorporation Assay in MCF-7 Cells

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MCF-7 cells at 30-50% confluence were cultured overnight in 25 cm² flasks. After 24 h of serum starvation, cells were treated with cytokines for 12 h or with SCM during 5, 7 and 10 days. After treatment, cells were labelled with 30 μM BrdU (Eurobio) for 25 min, washed and permeabilized with 100% ethanol. After treatment with pepsin and HCl 2 N to denature the DNA, samples were incubated with primary anti-BrdU and secondary anti-BrdU-FITC antibodies (Invitrogen). Samples were analyzed by flow cytometry.

Ortiz-Montero P., Londoño-Vallejo A, & Vernot J.P. (2017). Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line. Cell Communication and Signaling : CCS, 15, 17.

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Testicular EC Proliferation Assay

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5 × 10³ testicular ECs per well were plated on 8-chamber slide (LabTeK) coated with 0.1% gelatin. Busulfan (800 μM) or DMSO were added to each well 24 h after seeding and cultured for 72 or 96 h. Then, 10 μM BrdU (BD Pharmingen^TM) was added to media 72 h after busulfan treatment and then incubated for 2 h. Samples were stained with anti-BrdU (1:50; Invitrogen), anti-γ-H2AX (1:400; Millipore) and anti-cleaved caspase 3 (1: 400; Cell Signaling). BrdU⁺ cells in 10 random high-powered fields were counted and statistics were analyzed using GraphPad Prism. Each condition was performed in triplicate.

Bhang D.H., Kim B.J., Kim B.G., Schadler K., Baek K.H., Kim Y.H., Hsiao W., Ding B.S., Rafii S., Weiss M.J., Chou S.T., Kolon T.F., Ginsberg J.P., Ryu B.Y, & Ryeom S. (2018). Testicular endothelial cells are a critical population in the germline stem cell niche. Nature Communications, 9, 4379.

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Immunohistochemical Detection of Cell Proliferation

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Slides were prepared as above, and antigen retrieval was performed with citrate buffer (10mM Na citrate, pH 7) for 3 hours at 70°C. Sections were circled with a hydrophobic barrier pen (Vector Laboratories, Burlingame, CA) and blocked with an endogenous peroxidase / alkaline phosphatase inhibitor (Bloxall, Vector Laboratories). After washing with PBST (PBS + 0.1% Tween-20), they were incubated for 1 hour at RT in blocking solution (5% normal goat serum + PBST). To visualize cellular proliferation, sections were incubated with primary antibodies or IgG control overnight at 4°C: PCNA (#931143, Invitrogen, Carlsbad, CA), anti-BrdU (#933943, Invitrogen), anti-Ki67 at 1:100 (Abcam, Cambridge MA), Vectastain Elite ABC rabbit IgG kit (Vector Laboratories) and DAB Chromogen (Vector Laboratories).

Ackerman J.E., Bah I., Jonason J.H., Buckley M.R, & Loiselle A.E. (2017). Aging Does Not Alter Tendon Mechanical Properties During Homeostasis, but does Impair Flexor Tendon Healing. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 35(12), 2716-2724.

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Antibody Detection in Cell Study

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The following antibodies were used in this study: anti-α-smooth muscle actin, anti-BrdU (Invitrogen), anti-annexin V (Abcam), anti-p-Thr287 CaMKII (Cell Signaling Technology), anti-p47, anti-catalase, anti-SOD1, anti-p22, anti-GAPDH (Santa Cruz), anti-Nox1 (BD Biosciences), anti-SOD2 (Calbiochem) and anti-Nox4 (Epitomics). The generation of the anti-CaMKII [13 (link)] and anti-ox-Met 281/282 CaMKII [9 (link)] antibodies was described previously.

Zhu L.J., Klutho P.J., Scott J.A., Xie L., Luczak E.D., Dibbern M.E., Prasad A.M., Jaffer O.A., Venema A.N., Nguyen E.K., Guan X., Anderson M.E, & Grumbach I.M. (2014). Oxidative activation of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates vascular smooth muscle migration and apoptosis. Vascular pharmacology, 60(2), 75-83.

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Immunocytochemical Analysis of ARPE-19 Cells

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ARPE-19 cells were treated with 10 μM BrdU (#B5002; Sigma-Aldrich, USA). Then, the cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with PBS containing 0.1% Triton X-100 (PBS-T) for 15 min. The cells were treated with 2 N HCl at 37°C for 30 min for denaturation and washed twice with 0.1 M borate buffer for neutralization. Mouse RPE tissues or ARPE-19 cells were blocked with 3% BSA in PBS-T (blocking solution) for 1 h and incubated with anti-BrdU (#MA3-071; Invitrogen, USA), anti-ZO-1 (#61-7300; Invitrogen), anti-cleaved caspase-3 (#9661; Cell Signaling Technology), anti-lamin A/C (LMNA, #sc-376248; Santa Cruz Biotechnology), anti-p53, anti-p21, anti-p16 (#sc-1661; Santa Cruz Biotechnology), or anti-Ki67 (#ab15580; Abcam, UK) antibodies in blocking solution at 4°C overnight. The samples were washed with PBS, and a fluorescent dye-conjugated secondary antibody (#A-11029, #A-21424, #A-21429, #A-11034; Thermo Fisher Scientific) was applied and incubated for 2 h. Afterward, the samples were treated with Hoechst 33342 (#H3570; Invitrogen) in PBS for 15 min to stain the nuclei. The samples were mounted with Aqua-Poly/Mount (#18696-20; Polysciences, USA). Images were captured with a confocal microscope (#LSM 900; Carl Zeiss).

Ryu W., Park C.W., Kim J., Lee H, & Chung H. (2023). The Bcl-2/Bcl-xL Inhibitor ABT-263 Attenuates Retinal Degeneration by Selectively Inducing Apoptosis in Senescent Retinal Pigment Epithelial Cells. Molecules and Cells, 46(7), 420-429.

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