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5 protocols using risedronate

1

Characterization of OPG-deficient Mice

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OPG–/–mice (genetic background C57BL/6) [8 (link)] were purchased from CLEA Japan (Tokyo, Japan). OPG–/–mice were bred with WT mice (strain C57BL/6) obtained from Japan SLC (Shizuoka, Japan). Twelve-week-old male OPG–/– and OPG+/+ littermates (WT) were used in this study. The number of mice in the respective group was determined according to our previous studies [11 (link), 24 (link)]. All procedures for animal care were approved by the Animal Management Committee of Matsumoto Dental University (permit number: 267) and performed accordingly. W9 peptide [29 (link)] was provided by Oriental Yeast (Tokyo). Risedronate was obtained from LKT laboratories (St. Paul, MN). Other chemicals and reagents were of analytical grade.
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2

Osteoclast Differentiation Reagents

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Alendronate and risedronate were purchased from LKT laboratories (St. Paul, MN, USA). Minodronic acid was supplied by Astellas Pharma (Tokyo, Japan). Bafilomycin A1 was obtained from Wako Pure Chemical Industries (Osaka, Japan). Reagents for TRAP staining were purchased from Sigma Chemical (St. Louis, MO, USA). Alpha-modified Eagle's medium (α-MEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA).
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3

Risedronate Taste Solution Preparation

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Risedronate (LKT Laboratories, St Paul, MN, USA) was dissolved in vehicle (0.9% NaCl). The drug concentrations were adjusted to provide the appropriate dose in a total volume of 1.0 mL. The dose of Risedronate administered was based on that reported previously43 (link).
The following taste solutions were used in this study: NaCl, HCl, KCl, sucrose (with or without QHCl), QHCl, NH4Cl (all purchased from Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and MPG (Sigma-Aldrich, St Louis, MO, USA). All taste solutions were dissolved in distilled water (DW)52 (link).
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4

Mitochondrial Redox Modulation Protocol

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Risedronate was obtained from LKT Laboratories, Inc. (St. Paul, MN). QD powder was purchased from Seishinshouakudo (Tokyo, Japan), which imports QD from China. Cell Counting Kit-8 (CCK-8), diphenyl-1-pyrenylphosphine (DPPP), and MitoRed were purchased from Dojindo (Kumamoto, Japan). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from LABOTEC Co., Ltd. (Tokyo, Japan) and β-nicotinamide adenine dinucleotide (NADH), d-glutamic acid, and succinic acid were purchased from Sigma-Aldrich (St. Louis, MO). Malic acid, indirubin, and indigo were purchased from Wako Pure Chem. Ind., Ltd. (Osaka, Japan) and MitoTracker Green FM was purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).
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5

Quantitative Analysis of Risedronate

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Chemicals Risedronate and 1-octyltriethylammonium phosphate as an ion-pairing (IP) reagent were obtained from LKT Laboratories (St. Paul, MN, U.S.A.). Ammonium dihydrogen phosphate, calcium chloride, 1 M hydrochloric acid, and 1 M sodium chloride were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium salt (EDTA/2 Na), as a chelating agent, and tetrabutylammonium bromide were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Methanol was obtained from Kanto Chemical Industries (Tokyo, Japan). 2-Pyridylacetic acid hydrochloride, as an internal standard (IS), etidronate (ca. 60% in water, ca. 4.2 mol/L), and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), used as a calcium scavenger, were obtained from Tokyo Chemical Industry (Tokyo, Japan). OASIS ® HLB cartridges (Waters Corporation, Milford, MA, U.S.A.) were used for solid-phase extraction. Ultrapure water was generated using a Millipore Milli-Q Plus system (Merck Millipore, Billerica, MA, U.S.A.).
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