Anti brdu
Manufactured by BD
Sourced in United States, Germany
Anti-BrdU is a laboratory reagent used for the detection of bromodeoxyuridine (BrdU) in biological samples. It is a monoclonal antibody that specifically binds to BrdU, a synthetic analog of the DNA base thymidine that is incorporated into the DNA of dividing cells. This allows for the identification and quantification of cells undergoing DNA synthesis and cell division.
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Lab products found in correlation
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Bromodeoxyuridine (brdu), by BD (8 mentions)
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Hrp conjugated matched secondary antibodies, by Jackson ImmunoResearch (3 mentions)
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Ab6326, by Abcam (2 mentions)
Brdu flow kit protocol, by BD (2 mentions)
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92 protocols using anti brdu
1
BrdU and DAPI Staining Protocol
2
Antibody Sources for Cellular Assays
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Antibodies were purchased from the following sources: p21, PCNA, and anti-HA from Santa Cruz Biotechnology; PR-Set7 and Cdt1 from Cell Signaling Technologies; α-tubulin, GAPDH, and GST from Sigma; anti-BrdU from BD Biosciences; anti-phospho-Histone H2A.X (Ser139) from EMD Millipore; anti-HA from Roche Life Sciences; anti-UBCH8 and anti-UBE2G1 from Protein Tech; and anti-UBE2G2 from Thermo Scientific. Antibodies to human Cdt1 (Cook et al. 2004 (link)) and Cdt2 (Abbas et al. 2008 (link)) have been described previously. Alexa fluor 488, Rhodamine Red-X, and Cy5 donkey secondary antibodies for immunofluorescence microscopy were obtained from Jackson ImmunoResearch Laboratories.
3
Quantifying Cell Proliferation and Apoptosis in Mouse Lung
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Cell Proliferation Assay was performed as previously described [18 (link)]. Briefly, 5-bromo-2-deoxyuridine (BrdU) was administered 6 hours prior to tissue collection (75mg/kg, i.p.). Mouse lung cryosections (5μm) were stained overnight with anti-BrdU (1:5 dilution, BD Biosciences, USA) at 4°C then incubated with Alexa FITC-conjugated secondary antibody (1:200 dilution, Life Technologies, USA) for 30 minutes at room temperature. Lung vascular endothelial cells were immunostained with anti-vWF (1:300 dilution) and anti-CD31 (1:40 dilution) antibodies for 1 hour at room temperature. Sections were incubated with Texas Red-conjugated secondary antibody (1:700 dilution) for 30 minutes at room temperature. The nuclei were counterstained with DAPI. Two sections per lung tissue and 10 fields (20X) per section were quantified blindly.
Apoptosis was assessed using the In Situ Cell Death Detection Kit according to manufacturer’s instructions and anti-vWF and anti-CD31 antibodies were used to immunostain endothelial cells (ECs) as described above. Two sections per lung tissue and 10 fields (20X) per section were quantified blindly.
Apoptosis was assessed using the In Situ Cell Death Detection Kit according to manufacturer’s instructions and anti-vWF and anti-CD31 antibodies were used to immunostain endothelial cells (ECs) as described above. Two sections per lung tissue and 10 fields (20X) per section were quantified blindly.
4
Cell Cycle Analysis via BrdU and PI
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For dynamic cell cycle analysis (BrdU-incorporation) cells were pulsed with 10 μM BrdU for 1 h and stained with anti-BrdU according to a standard protocol (BD Biosciences). For static cell cycle analysis, cells were stained with propidium iodide (25 mg/ml PI; 100 µg/ml RNAse in PBS; Sigma-Aldrich, Heidelberg, Germany). Cell cycle distribution and apoptosis was determined by PI or two-dimensional (fluorescein and PI) flow cytometry. Analysis of DNA content occurred in the linear mode, apoptosis measuring in the logarithmic mode of the FL2 channel. Results were quantified using FlowJo software (Tree Star Inc., Ashland, OR). Cells with DNA content <2n were considered apoptotic (sub-G1).
5
BrdU Incorporation Assay for Cell Proliferation
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Cells (1–5 × 106 cells in 10 ml) were labeled with 20 µM BrdU for 20 min and collected. The cells were washed with 10 ml ice-cold PBS and fixed in 70% ethanol. After washing with 1 ml 1% BSA in PBS, the samples were incubated in 1 ml of 4 N HCl with 0.5% Triton X-100 at room temperature for 30 min and washed with 1 ml of 1% BSA in PBS three times. The cells were treated with anti-BrdU (BD) and stained with FITC-labeled anti–mouse IgG (Jackson ImmunoResearch). DNA was stained with 1 ml of 10 µg/ml propidium iodide in 1% BSA in PBS at 4°C overnight. The stained cells were applied to Guava easyCyte (Merck). Obtained data were analyzed with InCyte software (Merck).
6
Antibody Characterization for Cell Signaling Assays
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Antibodies used in this study are as follows. Anti-human Claspin was generated against the human recombinant Claspin with aa896–1,014 produced in E. coli. Anti-Chk1 phospho-S345 (#2348), anti-Chk1 phospho-S317 (#2344), anti-p44/42 MAPK (Erk1/2) (#4695), anti- SAPK/JNK (#9252), p38 MAPK (#8690), anti-p38 MAPK T180/Y182 (#4511), anti-p44/42 MAPK (Erk1/2) T202/Y204 (#4370), anti-SAPK/JNK T183/Y185 (#4668), Caspase-9 (#9508), Cleaved Caspase-3(#9661), and Mcl-1 (#5453) were obtained from Cell Signaling. Anti-α Tubulin (sc23948), anti-MCM2 (sc-9839), and anti-Chk1 (sc-8408) were obtained from Santa Cruz. Anti-phospho-H2A.X S139 (06-536) was purchased from Merck. Anti-BrdU (Ab6326) was purchased from Abcam. Anti-ATR phospho-T1989 (GTX128145) was purchased from GeneTex. Anti-BrdU (555627) was purchased from BD Pharmingen. Anti-H2A.X phospho-S139 (613402), anti-Rat IgG Alexa Fluor 555 (405420), and FITC-Anti-BrdU (364104) were purchased from Biolegend. RPA32 phospho-S4/S8 (A300-245A) and anti-MCM2 S53(A300-756A) were purchased from Bethyl. Anti-Mouse IgG Alexa Fluor 488 (A-11017) was purchased from Invitrogen. Goat Anti-Rabbit IgG HRP (111-035-003) and Goat Anti-Mouse IgG (115-035-003) were purchased from Jackson ImmunoResearch Laboratory.
7
Analyzing Cell Cycle and Proliferation
8
Comprehensive Pancreatic Tissue Analysis
9
Quantifying Retinal Endothelial Cell Proliferation
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To label proliferating cells in P5 retinas, BrdU (50 mg/kg, ip, Roche, #10280879001) was injected 4 h before sacrifice. Eyeballs were fixed in 4% PFA for 1 h and dissected retinas were rinsed briefly in water and incubated in 2N HCL for 2 h. After three washes with PBS, retinas were stained with Isolectin B4 in PBlec overnight at 4°C. Retinas were blocked and permeabilized in 10% goat serum (GeneTex, # GTX73206) and 0.25% Triton X‐100 in PBS for 6 h at 4°C and subsequently incubated with the primary antibodies anti‐BrdU (1:50, BD Biosciences, #347580) and anti‐ERG (1:500, Abcam, #ab110639) in blocking buffer overnight at 4°C. Secondary detection was performed with Alexa Fluor‐coupled secondary antibodies in blocking buffer for 6 h at 4°C. Retinas were flat‐mounted and analyzed as described in the previous section. Proliferating BrdU‐positive endothelial cells were normalized to the total number of ERG‐positive endothelial cells in 388 µm × 388 µm areas at the proliferating front.
10
Cell Proliferation Analysis by BrdU and Histone Modifications
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For BrdU incorporation analysis, MEFs were pulse-labeled for 1 h with 100 μM BrdU (BD Pharmingen, 550891) before collection. Collected cells were fixed in –20 °C cold 70% ethanol and washed in blocking buffer (PBS-bovine serum albumin (BSA) 1%; BSA Sigma-Aldrich, A7906). For non-BrdU analysis, cells were permeabilized with PBS-1% BSA-0.5% Triton X-100, whereas, for BrdU analysis, cells were treated with 2 n HCl/0.5% Triton X-100 and neutralized with 0.1 m sodium tetraborate, pH8.5. Cell staining were performed using anti-BrdU (1:500; BD Pharmingen, 555627, clone 3D4), pS10-histone H3 (1:100; Cell Signaling Technology [CST] #9701) or pS139-histone H2AX (1:500; CST #9718) followed by their incubation with secondary antibody Alexa 647-conjugated goat anti-mouse (1:400; Invitrogen, A21235) or Alexa 647-conjugated donkey anti-rabbit (1:400; Invitrogen, A31573). Before analysis, cells were resuspended in PBS supplemented with 2.5 μg/ml propidium iodide (Merck, 537059) and 20 μg/ml RNase A (Sigma-Aldrich, R6513). Analysis of cells and BrdU incorporation was acquired on BD FACSCalibur or BD LSRII flow cytometers (BD Bioscience) and further analyzed using FlowJo 8.8.7 software.
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