Npc1−/− mice on the BALB/cJ background were generated by mating heterozygous animals. Genotypes were identified from tail DNA by PCR as described (Loftus et al., 1997 (link)). Treatment with HPBCD (Sigma Aldrich, Milan, Italy) was performed in 7-day-old mice by a subcutaneous injection of 4 g HPBCD/kg body weight. BrdU labeling was achieved by intraperitoneal injection with 5-bromodeoxyuridine (60 mg BrdU/g body weight, Sigma Aldrich Inc., Milan, Italy). Mice were maintained in our animal facility in accordance with Sapienza University guidelines for the care and use of laboratory animals. Experimental protocols and related procedures were approved by the Italian Ministry of Public Health.
5 bromodeoxyuridine
Manufactured by Merck Group
Sourced in United States
5-bromodeoxyuridine is a synthetic nucleoside analog that is structurally similar to the DNA base thymidine. It can be used as a molecular biology research tool for the detection of DNA synthesis and cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33258, by Merck Group (2 mentions)
Collagenase type 2, by Merck Group (2 mentions)
Hpbcd, by Merck Group (1 mentions)
Dmrb microscope, by Leica (1 mentions)
Colchicine, by Merck Group (1 mentions)
Giemsa, by Avantor (1 mentions)
Eukitt, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Percoll, by Cytiva (1 mentions)
Pancreatin, by Merck Group (1 mentions)
Collagen coated dishes, by BD (1 mentions)
Sprague dawley rats, by Orient Bio (1 mentions)
Dab tablet, by Fujifilm (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Fetal calf serum (fcs), by Sartorius (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Sodium fluoride, by Merck Group (1 mentions)
Nonidet p 40, by Merck Group (1 mentions)
Penicillin and streptomycin, by Sartorius (1 mentions)
14 protocols using 5 bromodeoxyuridine
1
Npc1 Gene Knockout Murine Model
2
Sister Chromatid Exchange Assay
3
Neonatal Rat Ventricular Cardiomyocyte Isolation
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NRVMs were obtained from ventricles of 1-day-old Sprague-Dawley rats (Orient Bio, Korea). Cardiomyocytes were isolated and cultured as described previously (Sadoshima et al., 1992 (link)). Briefly, minced heart ventricular tissues were digested with HEPES-buffered saline solution containing 0.1% collagen type II (Thermo Fisher Scientific) and 0.4% pancreatin (Sigma-Aldrich) at 37°C. Cardiomyocytes were further purified by Percoll (Amersham Pharmacia Biotech, USA) gradient centrifugation. Cardiomyocytes were plated on collagen-coated dishes (BD Biosciences, USA) and cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Hyclone, USA), 2 mM L-glutamine (Invitrogen, USA), and 100 μM 5-bromodeoxyuridine (Sigma-Aldrich) for 24 h.
4
Detection and Analysis of Apoptosis and Proliferation
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For detection of apoptotic cells, a TUNEL assay was carried out using an In Situ Cell Death Detection Kit, POD (Roche). FITC signals were detected in the apoptotic cells at E11.0. For analysis of cell proliferation, 5-bromodeoxyuridine (BrdU, Sigma B5002) was intraperitoneally injected into pregnant females (40 mg kg−1) at E11.0. Two hours later, embryos were collected and fixed with 4% paraformaldehyde in PBS and embedded in paraffin. Immunostaining was carried out as described elsewhere. Briefly, the deparaffinized sections (5 μm thickness) were treated with 0.3% H2O2, 2 N HCl, trypsin and reacted with monoclonal mouse BrdU antibody (Sigma clone B8434) and biotynylated mouse anti-IgG (Vector M.O.M. peroxidase Kit PK-2200). For colouring, a DAB tablet (Wako 049-22831) was used.
5
Cell Culture and Nucleic Acid Labeling
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Sodium fluoride (NaF), Hoechst 33258, 5-bromodeoxyuridine (BrdU), phytohemagglutinin (PHA), Nonidet P-40, sodium dodecylsulfate and aprotinin were obtained from Sigma Chemical Company (St Louis, MO, USA). Fetal calf serum (FCS), penicillin and streptomycin and l -glutamine were used from Biological Industries, Israel. Giemsa stain was obtained from BDH chemicals, UK.
Other chemicals used in this study were of analytical grade from reputed manufacturers.
Other chemicals used in this study were of analytical grade from reputed manufacturers.
6
Isolation of Rat Cardiomyocytes from Newborns
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Healthy newborn (approximately 1–3-day old) Sprague-Dawley rats were used to isolate rat cardiomyocytes following previously reported procedures (20 (link)). In brief, rats were sacrificed by cervical dislocation and the heart was removed. Then, the cardiac apex was obtained and rinsed twice with pre-cooled phosphate buffered saline (PBS). After being finely chopped, fragments of the cardiac apex tissues were digested with 0.25% trypsin (Gibco, USA) 5-6 times at 37°C. Next, cardiomyocytes were collected by filtration and centrifugation at 8000 g for 5 min at 37°C, and maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 5′-bromodeoxyuridine (0.1 mM, Sigma, USA). On day 3, in order to remove the growth medium, the cardiomyocytes were incubated with serum-free DMEM for the next experiments.
7
Molecular Biology Reagents and Assays
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Dox and 5-bromodeoxyuridine were purchased from Sigma–Aldrich. Primers for real-time RT–PCR were synthesized by Integrated DNA Technologies, Inc. The Dual-Luciferase Reporter Assay System was purchased from Promega. Recombinant human EPO (carrier free) was purchased from BioLegend (catalog no.: 587102). All other cell culture reagents were purchased from Life Technologies or Sigma–Aldrich.
8
Isolation and Characterization of Cardiac Cell Types
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Dulbecco's modified Eagle's medium (DMEM)/F12 (1:1) and foetal bovine serum were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Trypsin, type II collagenase and 5-bromodeoxyuridine were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). DH5α competent cells were purchased from Tiangen (Beijing, China). Specific rabbit monoclonal antibodies against hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4), which is a marker for SAN, connexin 43 (COX43), which is a common connexion between cardiac cells, cardiac troponin I (cTnI), which is a marker for NRVMs, α-striated actin (α-SA), which is a marker for NRVMs, and GAPDH were purchased from Abcam (Cambridge, MA, USA); antibodies for vimentin, which is a marker for CFs, and COX-45, which is another common connexion between cardiac cells, were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), respectively; and antibodies for myosin heavy chain (MHC), which is a marker for NRVMs, and α-striated muscle actin (α-SMA), which is a marker for cardiac myofibroblasts (CMFs) were purchased from Wuhan Sanying Biotechnology (Wuhan, China) and Wuhan Tiandeyue Biotechnology (Wuhan, China), respectively.
9
Analyzing Neurogenesis via BrdU Labeling
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For the analysis of neurogenesis, the animals received intraperitoneal injections of 5-bromodeoxyuridine (BrdU, Sigma-Aldrich) 50 mg/g of body weight at a concentration of 10 mg/ml in sterile saline for 4 consecutive days.
10
Isolation and Culture of Neonatal Rat Ventricular Myocytes
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NRVMs were used for all the experiments in vitro in this study, and they were isolated from 1- to 2-day-old Sprague–Dawley rats. As reported previously (34 (link)), tissues were mechanically crushed in cold PBS and then transferred into 0.125% trypsin (in PBS). Digestion, for one cycle, involved a 15-min incubation at 37 °C and centrifugation of the suspension, then resuspending precipitation in Dulbecco's modified Eagle's medium (Life Technologies) with 10% fetal calf serum (Life Technologies) and penicillin (100 U/ml)/streptomycin (100 μg/ml) (Life Technologies). Four cycles later, the fibroblasts were removed from the resuspension by differential plating at 37 °C in 5% CO2 for 90 min. NRVMs were distributed into corresponding dishes, and the media were renewed after 24-h incubation with 100 μM 5-bromodeoxyuridine (Sigma–Aldrich).
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