For 5 days before the extraction procedure, the rats were fed a diet containing β-aminopropionitrile (β-APN; Sigma-Aldrich, St Louis, MO, USA) at a rate of 0.4% β-APN per gram of chow to facilitate subsequent extraction of the maxillary first molars. Under general anesthetic via an intramuscular injection of alfaxalone (Alfaxan®, 0.1 ml/100 g), the left and right maxillary first molars of all rats (n = 48) were extracted, and bleeding was controlled. An intramuscular injection of gentamicin (3 mg/kg; DaeSung Microbiological Labs, Uiwang, Korea) and a subcutaneous injection of 1% ketoprofen (0.3 ml/kg; Uni Biotech, Chungnam, Korea) were administered to prevent infection and to relieve pain, respectively, for 3 days after the extraction.
β apn
Manufactured by Merck Group
Sourced in United States
β-APN is a laboratory product offered by the Merck Group. It serves as a reagent used in various analytical and research applications. The core function of β-APN is to facilitate specific chemical reactions and analyses, though its precise intended use should not be extrapolated beyond this factual description.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca kit, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
4 aminophenylmercuric acetate, by Merck Group (1 mentions)
Pd173074, by Selleck Chemicals (1 mentions)
Compound c, by Selleck Chemicals (1 mentions)
β aminopropionitrile, by Merck Group (1 mentions)
Zoletil 50, by Virbac (1 mentions)
Rpmi 1640 glutamax, by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Tgf β, by R&D Systems (1 mentions)
6 protocols using β apn
1
Maxillary Molar Extraction in Rats
2
Measurement of VEGF Binding Affinity
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If not otherwise indicated, chemicals were purchased from Sigma Aldrich (Munich, Germany) or Carl Roth (Karlsruhe, Germany). βAPN was purchased from Sigma Aldrich. Protein concentrations were determined with the Pierce BCA Kit (Thermo Fisher, Rockford, IL, USA) using a 30-min incubation time at 60 °C. The anti-VEGF antibody mG6-31 and a control IgG (anti-Ragweed) were provided by Genentech (San Francisco, CA, USA).
3
Adipocyte Isolation and Genetic Manipulation
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Primary adipocytes were isolated and cultured as per a previously described method [56 (link)]. In experiments involving the administration of Ad-Col15a1 or sh-Col15a1 (24 h or 48 h at the titer of 1 × 109 IFU/mL), AMPK inhibitor (Compound C, Selleck, Houston, Texas, USA), mTORC1 inhibitor (rapamycin, Selleck), MMP agonist (4-Aminophenylmercuric acetate, Sigma–Aldrich, St. Louis, Missouri, USA), FGFR1 inhibitor (PD173074, Selleck), LOX inhibitor (β-APN, Sigma–Aldrich), or FGF2 recombinant protein (50037-M07E, Sino Biological, Beijing, China), cells were allowed to adhere for 48 h before treatment. The medium was subsequently replaced, and adipocyte petri dishes were treated with the indicated reagents. Cells were assessed by RT-PCR and western blot or fixed with 4% paraformaldehyde and stained for markers of interest.
4
Avoidance of Traumatic Tooth Extraction
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For the surgical intervention, the animals were anesthetized by administering Zoletil 50 (Virbac Lab, Carros, France) intramuscularly at a dose of 100–150 mg/kg and underwent a 5-day treatment with 0.4% β–aminopropionitrile (β-APN; Sigma-Aldrich, St. Louis, MO, USA) to avoid traumatic extraction [15 (link)]. Forty bilateral maxillary first molars with complete root formation were extracted from 20 animals. Preoperatively, the oral cavity of the animals was cleaned with 2% chlorhexidine solution. Extraction of the maxillary first molars was performed gently using sterile extraction forceps. After excluding any roots that fractured during extraction, the allotment of 10 teeth per group was maintained by including additional experimental animals. The extracted teeth were exposed to a dry environment for 5 minutes at room temperature or preserved in HBSS solution for 60 minutes; subsequently, the teeth and sockets were cleaned with saline prior to replantation. After replantation, a single dose of 20,000 IU of penicillin G (Alvogen potassium penicillin G, Alvogen, Seoul, Korea) was administered intramuscularly in all animals, and they were fed a soft diet for 7 days [7 (link)]; euthanasia was performed 8 weeks after replantation (Figure 1 ).
5
Metastatic Breast Cancer Cell Culture
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Human Hs578T and MDA-MB-231 breast cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA) and authenticated using short tandem repeat analysis. The human MDA-B02 breast cancer cell line (B02) is a subpopulation of the MDA-MB-231 cell line that was selected for its high and selective efficiency to metastasize to bone in mice [13] (link). MDA-MB-231 and B02 cells were cultured in DMEM medium containing 1.5 g/l glucose (Invitrogen). Hs578T, MCF-7 and T47D cells were cultured in RPMI-1640-Glutamax (Invitrogen). Culture media were supplemented with 10 % (v/v) FBS, and cultured cells maintained in a CO2 incubator at 37 °C. When specified, tumor cells were cultured with 350 μM βAPN (Sigma).
6
Fibroblast Repopulation of Decellularized Lung Matrices
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Decellularized lung slices from both non-diseased and IPF subjects were washed three times in sterile DMEM solution and were subsequently added to a suspension of 5.0×105 fibroblasts ml− 1 in growth media at a ratio of 1 matrix:2 ml of cell solution (n=5 non-IPF, n=5 IPF cell lines and n=1 no-cell control) up to a maximum of 25 ml of solution in a 50 ml Falcon tube. The Falcon tubes containing the matrices and cell suspension were mounted on a suspension mixer (Selby, ThermoFisher Scientific, Waltham, MA, USA) and constantly rotated at low speed (approximately one rotation every 20 s) at 37°C for 24 h. The reseeded ECMs were placed in separate wells of a 48-well plate with fresh media [0.01% DMSO +0.1% BSA ±10 ng ml− 1 TGF-β (R&D Systems, MN, USA) ±300 nM Compound A or 0.1 mM β-APN (Sigma)] and incubated at 37°C and 5% CO2. The reseeded decellularized matrices were collected at day 7; half were fixed in 4% paraformaldehyde solution for 24 h and paraffin embedded, whereas the other half were fresh frozen in optimal cutting temperature (OCT) compound and stored at −80°C.
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