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5 protocols using ketoprofen

1

Anti-inflammatory Compound Screening in Macrophages

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BMDMs were treated with with 0.125 – 0.5 mM indomethacin (Sigma-Aldrich), 0.25 – 1 mM (S)-ibuprofen (Cayman Chemical), 0.125 – 0.5 mM ketoprofen (Sigma-Aldrich), 0.25 – 1 mM Etodolac (Cayman Chemical), or 0.25 – 1 mM naproxen (Cayman Chemical), 0.25–1 mM rofecoxib, 5 mM sulforaphane (Cayman Chemical), 100 μM dimethylfumarate. NSAIDs were diluted in 1% DMSO. Cells were pretreated with NSAID for 1 hour prior to stimulation with 10 ng/mL LPS (L2880, Sigma-Aldrich). LDH assay performed according to manufacturer’s instructions (601170, Cayman Chemical). Reactive oxygen species (ROS) measured using H2DCFDA (D399, ThermoFisher) as described previously (Kavian et al., 2018 (link)). Sodium arsenite (Sigma-Aldrich), naphthalenedione (DMNQ, Cayman Chemical) and phenazine methosulfate (PMS, Cayman Chemical) at the indicated doses were used to induce ROS in BMDM.
MDMs were treated with indicated concentrations of indomethacin (Sigma-Aldrich), ketoprofen (Cayman Chemical), or sulforaphane (Cayman Chemical) for indicated durations.
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2

Formulation and Dissolution of Oral Capsules

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Vcaps® Enteric capsules, size 0, were obtained from Lonza Capsules and Health Ingredients (Morristown, NJ, USA). Trimethoprim (≥98% purity) and ketoprofen (≥98% purity) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Acetaminophen (98–102% pure powder) and atenolol (≥98% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Microcrystalline cellulose (MCC) (MICROCEL® MC-101) and mannitol (PEARLITOL® 100 SD) were received as a gift from Roquette (Keokuk, IA, USA). Sodium dihydrogen phosphate monohydrate, sodium hydroxide and 5 M HCL were purchased from VWR (Radnor, PA, USA). Deionized water was used throughout.
Dissolution accessories were purchased from Quality Lab Accessories (Telford, PA, USA), including: 200 mL vessels (175 mm height, 45 mm internal diameter) and mini-paddles (300 mm length, 9 mm height of paddle blade), USP compliant Japanese Pharmacopoeia sinker baskets and ultra-high-molecular-weight polyethylene (UHMPE) 10 μm porous filters.
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3

Receptor Binding Assay Protocol

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Corticosterone, picrotoxin, and ketoprofen were purchased from Cayman Chemical. FITC-AffiniPure Donkey Anti-Mouse IgG (H + L) and AlexaFluor-594-IgG Fraction Monoclonal Mouse Anti-Biotin were purchased from Jackson ImmunoResearch Laboratories. TTX was purchased from the Research Institute of the Aquatic Products of Hebei. All other regents and drugs were purchased from Sigma Millipore, Tocris Bioscience, or BBI Life Sciences.
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4

Lipid Extraction and Quantification

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Ketoprofen, 2-AG, PGE2, AA, d5-2-AG, d4-PGE2, and d8-AA were obtained from Cayman Chemical (Ann Arbor, MI, USA). Mass spectrometry-grade chloroform, methanol, acetonitrile, dimethylsulfoxide (DMSO), and water were obtained from Thermo Fisher Scientific (Waltham, MA, USA).
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5

Standardized Mouse RYGB Surgery Protocol

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RYGB
and sham surgeries
were performed under inhaled 3–5% isoflurane anesthesia as
previously described (“RYGB” procedure).66 (link) Mice which did not achieve a body weight of
at least 33 g on the day before surgery were excluded from the study.
Prior to surgery, mice were fasted for approximately 12 h. On the
morning of surgery, the mice received 0.03 mg of buprenorphine analgesic
(Patterson Veterinary via Hospira Inc., Nashville, TN) and 0.1 mL
of 0.9% saline (intraperitoneal). Animals were administered 0.017
mg of ketoprofen (Cayman Chemical, Ann Arbor, MI) once/day as needed
for 1–2 days following surgery and weekly iron dextran injections
(10 mg/kg; Durvet, Inc., Blue Springs, MO) to prevent anemia.
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