.4008, Carrigtwohill, Ireland). Formic acid, ethyl acetate, trifluoroacetic acid, ammonium acetate, and L-arginine hydrochloride had been bought from Macklin Biochemical Co., Ltd (F809712, E809174, T818778, A801000, L800291, Shanghai, China). The 2-chloro-L-phenylalanine and dansyl chloride were bought from Yuanye Bio-Technology Co., Ltd (B25643, CYP11 Inhibitor manufacturer S19248, Shanghai, China). The 1,4-Butane1,1,two,2,three,3,four,4-d8-diamine was bought from Toronto Investigation Chemical compounds (D416027, Canada). Chloroform, sodium carbonate, sodium bicarbonate, and acetone were bought from Lingfeng Chemical Reagent Co. (Shanghai, China). Ornithine analytical regular was bought from Solarbio (SO8470, Beijing, China). RPMI-1640 medium without arginine was bought from Sigma-Aldrich (R1780, USA). Certified fetal bovine serum, RPMI 1640 medium, and dialyzed fetal bovine serum have been purchased from Biological Industries (05-065-1A, 04-002-1A, 04-011-1b, Kibbutz Beit Haemek, Israel). Cell counting kit (CCK-8) was purchased from Yeasen Biotechnology Co., Ltd. (40203ES76, Shanghai, China).Animal ProtocolsAll animal procedures were carried out in accordance with government-published suggestions for the Care and Use of laboratory Animals and authorized by the Institutional Ethics Overview Boards of Nanjing University of Chinese Medicine. The experimental mice (C57BL/KaLwRij, six weeks, 180 g) had been purchased from the institute of model animals of Nanjing University. The experimental mice were housed within the SPF laboratory animal center of Nanjing University of Chinese Medicine with 155 ambient temperature and totally free access to meals and water. Immediately after 1 week of adaptive feeding, animal experiments have been began. 5TMM3VT murine D1 Receptor Inhibitor Storage & Stability myeloma cells (106) were injected through the tail vein into 6-week-old C57BL/ KaLwRij mice. The mice were divided into 4 groups as follows: model handle (C, only modeling, n=10) group, bortezomib (V, n=10) therapy group, acupuncture (A, n=9) remedy group, and VA (n=8) remedy group. A single day later, mice in 3 treatment groups have been treated by unique approaches, containing intraperitoneal injection of 1.2 mg/ml V twice per 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, 2 mA) 3 occasions per week until all of the mice were dead.Serum Sample Collection Procedures Chemical compounds 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 throughout the experimental period. Blood samples of week four were mixed within the clean Eppendorf tubes, stored on ice for 2 h, and centrifuged (five,000 rpm, ten min) at four . The blood was collected for separation of serum. Subsequently, the 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 had been thawed around the ice. An aliquot of 45 ml serum sample was precipitated by adding 135 ml acetonitrile containing internal requirements (IS) [2-Chloro-L-Phenylalanine (plasma sample-acetonitrile: IS=2,000:1)], vortex for 30 s, sonicating for 10 min at 4 , then stayed for 3 h on ice. Precipitated protein was removed by centrifugation (13,000 rpm, ten min) at 4 . Subsequently, 153 ml supernatant was transferred to glass inserts of LC-MS vials and stored