The chemicals used were 100 mM stock solution of BrdU (Merck, B5002) and 10 mM stock solutions of PARG inhibitor (PDD0017273, Tocris, 5952; Merck, SML1781), PARP inhibitors (KU0058948, Axon Medchem, Axon 2001; Olaparib, ApexBio, A4154), FEN1 inhibitor (UOS-33991; compound 17 in ref. 52 (link) and synthesized in-house as described in ref. 11 (link)), REV1 TLS inhibitor (JH-RE-06, Axon Medchem, Axon 3002) and EdU (Cambridge Bioscience, CAY20518) were prepared in dimethyl sulfoxide (DMSO). CldU (Merck, C6891) and IdU (Merck, I7125) were dissolved directly in culture medium at a final concentration of 2.5 mM, and thymidine (Merck, T1895) in culture medium at 200 mM. 1 mCi per ml 3H-Thymidine (PerkinElmer, NET027W005MC) in 2% ethanol and 99% MMS were added directly to culture medium to a final concentration of 2 µCi per ml and 0.01%, respectively.
B5002
Manufactured by Merck Group
Sourced in United States, United Kingdom, Canada
The B5002 is a laboratory centrifuge designed for general-purpose use in research and clinical settings. It features a compact and durable construction, accommodating a range of sample sizes and volumes. The centrifuge can achieve speeds up to 6,000 rpm, enabling efficient separation of samples. Key specifications and capabilities are provided in the product documentation.
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Lab products found in correlation
Bromodeoxyuridine (brdu), by Merck Group (8 mentions)
Ab6326, by Abcam (8 mentions)
I7125, by Merck Group (5 mentions)
B8434, by Merck Group (5 mentions)
Propidium iodide, by Merck Group (4 mentions)
C6891, by Merck Group (3 mentions)
B2531, by Merck Group (3 mentions)
A10044, by Thermo Fisher Scientific (3 mentions)
Rnase a, by Merck Group (3 mentions)
Tamoxifen, by Merck Group (3 mentions)
Facscanto 2, by BD (3 mentions)
Foxp3 transcription factor staining buffer set, by BioLegend (2 mentions)
True nuclear transcription factor buffer set, by BioLegend (2 mentions)
D4513, by Merck Group (2 mentions)
G7021, by Merck Group (2 mentions)
Specific anti brdu fitc monoclonal antibody, by BD (2 mentions)
En0531, by Thermo Fisher Scientific (2 mentions)
P3566, by Thermo Fisher Scientific (2 mentions)
Eclipse e800, by Nikon (2 mentions)
P4864, by Merck Group (2 mentions)
189 protocols using b5002
1
DNA Replication Inhibitors Screening
2
BrdU Incorporation Assay for Proliferation
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Forty-eight hours after transfection, the culture medium was changed to fresh medium supplemented with 10 µM of bromodeoxyuridine (BrdU) (B5002, Merck KGaA) and incubated for 30 min at 37°C. The cells were fixed with ice-cold methanol for 10 min, washed three times with PBS, and incubated with 2 M of HCl for 10 min at room temperature. After being washed, the cells were incubated with an Anti-BrdU antibody for 1 h at room temperature. Finally, the cells were incubated with fluorescent-labeled Alexa Fluor 488 and DAPI (1:200) and mounted using Fluoromount. The stained slides were scanned with 3DHistech Pannoramic Confocal scanner and analyzed using CaseViewer CellQuant 2.2 (3DHISTECH Ltd., Budapest, Hungary).
3
Fluorescent Nucleoside Tracking Assay
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4
BrdU Incorporation Assay
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Culture medium was replaced with fresh medium supplemented with 10 µM BrdU (B5002, Merck KGaA, Darmstadt, Germany) and was incubated for 30 min at 37 °C. Ice-cold methanol was used for 10 min to fix the cells. After washing with PBS for 3 × 5 min, cells were incubated with 2N HCl for 10 min at room temperature. Cells were again washed three times, then incubated with 0.2% Triton X-100 to improve the penetration of the Anti-BrdU antibody. After one hour of incubation at room temperature the cells were washed and labeled with Alexa Fluor 488 and DAPI (1:200). The cells were mounted using Fluoromount.
5
Hippocampal Cell Proliferation Analysis
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For analysis of cell maintenance in the hippocampal DG (Sun et al., 2015), BrdU (Merck, B5002) was dissolved in normal saline at a stock concentration of 10 mg/mL and was injected (50 mg/kg, i.p.) into mice once per day for 7 consecutive days. Mice were analyzed 14 days after the first 5′-bromo-2′-deoxyuridine (BrdU) injection (Zhou et al., 2018). To investigate EtOH-induced changes in cell proliferation, pulse BrdU (300 mg/kg, i.p.) was injected only once 2 hours before mice were sacrificed (Nixon et al., 2008). Quantification of BrdU+ cells within the subgranular zone (SGZ) and granule cell layer (GCL) was carried out as previously described (Zhou et al., 2018).
6
Analyzing Cell Proliferation and Fate in Mouse Embryonic Development
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For analysis of proliferation in vivo, pregnant mice were intraperitoneally injected with BrdU (50 mg/kg; B5002; Sigma-Aldrich) at different embryonic stages (E12, E14, E16, and E18) and sacrificed 30 min thereafter (Wu et al., 2017 (link)). For determination of the cell cycle exit, BrdU (100 mg/kg) was intraperitoneally injected into pregnant mice, and mice were sacrificed 18 h after injection. The cell cycle exit index was calculated as BrdU+Ki67−/total BrdU+ cells (the percentage of cells exiting the cell cycle). The length of S-phase of the cell cycle was calculated as BrdU+Ki67+/total Ki67+ cells.
For pulse-chase labeling of newborn cells, pregnant mice were intraperitoneally injected with BrdU (100 mg/kg) at E11.5, E14.5, and E15.5 and sacrificed at E18 or injected with BrdU at E16.5 and sacrificed at P2. Quantification of birth-dated neurons was performed by calculating the percentages of BrdU+ layer-specific neuronal marker+ cells/total layer-specific neuronal marker+ (Tbr1+, Ctip2+, or Cux1+) cells. Quantitation of birth-dated oligodendrocytes was performed by calculating the percentage of Olig2+BrdU+ cells/total Olig2+ cells. Quantification of birth-dated astrocytes was calculated as the number of GFAP+BrdU+ cells along the dorsolateral VZ.
To investigate NPC proliferation in vitro, cells were cultured for 4–5 h in NPC culture medium supplemented with 10 µM BrdU.
For pulse-chase labeling of newborn cells, pregnant mice were intraperitoneally injected with BrdU (100 mg/kg) at E11.5, E14.5, and E15.5 and sacrificed at E18 or injected with BrdU at E16.5 and sacrificed at P2. Quantification of birth-dated neurons was performed by calculating the percentages of BrdU+ layer-specific neuronal marker+ cells/total layer-specific neuronal marker+ (Tbr1+, Ctip2+, or Cux1+) cells. Quantitation of birth-dated oligodendrocytes was performed by calculating the percentage of Olig2+BrdU+ cells/total Olig2+ cells. Quantification of birth-dated astrocytes was calculated as the number of GFAP+BrdU+ cells along the dorsolateral VZ.
To investigate NPC proliferation in vitro, cells were cultured for 4–5 h in NPC culture medium supplemented with 10 µM BrdU.
7
BrdU and Tamoxifen Labeling in Mice
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Mice were injected with BrdU and Tamoxifen as previously described (Appel et al., 2018 (link)). Briefly, mice were injected with BrdU (Sigma, 10 mg/mL, B5002; i.p., 200 mg/kg body weight) 2 h before perfusion. Tamoxifen (10 mg/mL, Sigma, T5648) was prepared in corn oil (Sigma, C8267) mixed with ethanol (9:1 ratio). Mice were injected with 100 mg/kg Tamoxifen (i.p., daily for constitutive 5 days) for 4-week-old male mice and with 125 mg/kg Tamoxifen (i.p., every 12 h for 4 times) for 8-week-old male mice. Mice were perfused at 1 month after injection and 2 days after injection, respectively.
8
Adult Fish Cardiac Injury Models
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This work was performed with fully grown adult fish at the age of 12–24 months. Wild-type fish were AB (Oregon), transgenic fish lines were cmlc2:DsRed2-nuc [21 (link)] and cmlc2:EGFP [22 (link)]. The control and preconditioned animals for each experiment were siblings that were maintained in the same density and food conditions. Before every procedure, fish were anaesthetized with tricaine (Sigma-Aldrich). To perform thoracotomy, fish were placed ventral side up on a damp sponge and a small incision was made through the thorax skin with iridectomy scissors. The peritoneal injections were performed by injection of 2 µl of solution into the abdomen of the fish using a glass microcapillary connected to the Femtojet transjector (Eppendorf). The lipopolysaccharides (LPS; Sigma-Aldrich) and Zymosan (Sigma-Aldrich) were injected at a concentration of 10 mg ml−1 in Hank's solution (20 µg of immunogenic particles injected per fish). Cryoinjuries were performed as described previously [23 (link),24 ]. For bromodeoxyuridine (BrdU) incorporation experiments, the animals were maintained in 5 mg ml−1 BrdU (B5002; Sigma-Aldrich) for 7 or 30 days. During all treatments, fish were fed and solutions were changed every third day.
9
BrdU Labeling of Mouse Testes
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The BrdU labeling was done as described previously.26 (link) Briefly, mice were injected intraperitoneally with BrdU (50 μg g−1 body weight, B5002, Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS), then killed 2 h later. Testes were collected and fixed in 4% paraformaldehyde (PFA)/PBS solution.
10
Multiparametric Intracellular Staining Protocol
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For Ki67, DAPI, RELMα, BrdU and pHH3 staining, the eBioscience FoxP3/Transcription Factor Staining Buffer set (00-5523-00) or the BioLegend True-Nuclear Transcription Factor Buffer set (424401) was used. In brief, after surface staining, cells were fixed with 1x Fix Concentrate buffer in provided Fix Diluent for 30 minutes at 4 °C. Cells were then washed with FACS buffer and stored overnight. To permeabilize the cells, samples were washed with 1x Perm buffer diluted in water. Following blocking with 2% rat serum, samples were stained for 1 hour at RT, except for DAPI and secondary antibodies (20 minutes at RT), followed by washing in 1x Perm buffer and FACS buffer before flow cytometry.
For BrdU staining, mice were given 1 mg of BrdU i.p. (from BD kit, 552598, or Sigma, B5002) three hours before sacrifice as described12 (link). After sacrifice of mice and peritoneal lavage, samples were processed, fixed and stored overnight as for other intracellular antigens. BrdU-labelled cells were washed in 1x Perm buffer, incubated in DNase I (from BD kit, 552598 or Sigma, D4513) in 1x DPBS for 30 min at 37 °C, washed in 1x Perm buffer, blocked with 2% rat serum and stained for 1 hour at RT with α-BrdU antibody (BD, 552598), followed by washing in 1x Perm buffer and FACS buffer. Mice that did not receive BrdU were used as negative controls.
For BrdU staining, mice were given 1 mg of BrdU i.p. (from BD kit, 552598, or Sigma, B5002) three hours before sacrifice as described12 (link). After sacrifice of mice and peritoneal lavage, samples were processed, fixed and stored overnight as for other intracellular antigens. BrdU-labelled cells were washed in 1x Perm buffer, incubated in DNase I (from BD kit, 552598 or Sigma, D4513) in 1x DPBS for 30 min at 37 °C, washed in 1x Perm buffer, blocked with 2% rat serum and stained for 1 hour at RT with α-BrdU antibody (BD, 552598), followed by washing in 1x Perm buffer and FACS buffer. Mice that did not receive BrdU were used as negative controls.
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