.4008, Carrigtwohill, Ireland). Formic acid, ethyl acetate, trifluoroacetic acid, ammonium acetate, and L-arginine hydrochloride have been purchased from Macklin Biochemical Co., Ltd (F809712, E809174, T818778, A801000, L800291, Shanghai, China). The 2-chloro-L-phenylalanine and dansyl chloride were purchased from Yuanye Bio-Technology Co., Ltd (B25643, S19248, Shanghai, China). The 1,4-Butane1,1,two,2,3,three,4,4-d8-diamine was purchased from Toronto Research Chemical compounds (D416027, Canada). Chloroform, sodium carbonate, sodium bicarbonate, and acetone have been purchased from Lingfeng Chemical Reagent Co. (Shanghai, China). Ornithine analytical standard was purchased from Solarbio (SO8470, Beijing, China). RPMI-1640 medium devoid of arginine was bought from Sigma-Aldrich (R1780, USA). Certified fetal bovine serum, RPMI 1640 medium, and JAK3 Inhibitor site dialyzed fetal bovine serum had been bought from Biological Industries (05-065-1A, 04-002-1A, 04-011-1b, Kibbutz Beit Haemek, Israel). Cell counting kit (CCK-8) was bought from Yeasen Biotechnology Co., Ltd. (40203ES76, Shanghai, China).Animal ProtocolsAll animal procedures have been conducted in accordance with government-published recommendations for the Care and Use of Laboratory Animals and authorized by the Institutional Ethics Critique Boards of Nanjing University of Chinese Medicine. The experimental mice (C57BL/KaLwRij, 6 weeks, 180 g) were bought from the institute of model animals of Nanjing University. The experimental mice had been housed within the SPF laboratory animal center of Nanjing University of Chinese Medicine with 155 ambient temperature and cost-free access to meals and water. Immediately after 1 week of adaptive feeding, animal experiments were started. 5TMM3VT murine myeloma cells (106) had been injected through the tail vein into 6-week-old C57BL/ KaLwRij mice. The mice have been divided into 4 groups as follows: model manage (C, only modeling, n=10) group, bortezomib (V, n=10) therapy group, acupuncture (A, n=9) treatment group, and VA (n=8) therapy group. One particular day later, mice in 3 therapy groups were treated by various techniques, containing intraperitoneal injection of 1.2 mg/ml V twice a week or/and electroacupuncture (Model SDZ-II, Suzhou Medical Appliance Factory, Suzhou, China) stimulation of Hegu (29) and Zusanli (30, 31) points (2/100 Hz, two mA) three occasions per week till all the mice were dead.Serum Sample Collection Techniques Chemicals and ReagentsThe 5TMM3VT murine myeloma cells had been donated by Professor Wen Zhou in the State Essential Laboratory of Blood was taken from tail vein on Tuesday and Wednesday in the course of the experimental period. Blood samples of week 4 were mixed within the clean Eppendorf tubes, stored on ice for 2 h, and centrifuged (5,000 rpm, 10 min) at four . The blood was collected for separation of serum. Subsequently, the KDM3 Inhibitor Molecular Weight supernatants were transferred to clean Eppendorf tubes and stored at -80 .Frontiers in Oncology | frontiersin.orgNovember 2021 | Volume 11 | ArticleKe et al.Acupuncture and Bortezomib Benefit MMSample Preparation for LC-MS AnalysisAll serum samples were thawed around the ice. An aliquot of 45 ml serum sample was precipitated by adding 135 ml acetonitrile containing internal standards (IS) [2-Chloro-L-Phenylalanine (plasma sample-acetonitrile: IS=2,000:1)], vortex for 30 s, sonicating for ten min at four , and after that stayed for three h on ice. Precipitated protein was removed by centrifugation (13,000 rpm, 10 min) at four . Subsequently, 153 ml supernatant was transferred to glass inserts of LC-MS vials and stored
