Iv icpms 71a

AS solutions (0.15 M) in ultrapure water were analyzed for trace metals using a Thermo Scientific iCAP RQ ICP-MS operating in KED mode (He atmosphere). The analytical matrix is 2% vol/vol Omnitrace HNO₃ and 0.5% vol/vol Omnitrace HCl. The instrument was calibrated using a 43-element calibration solution (Inorganic Ventures IV-ICPMS-71A). A 6-element internal standard (IV-ICPMS-71D) was added to samples prior to quantitation. Quality control checks were performed using NIST-1643f and Inorganic Ventures IV-STOCK-50 reference solutions.

Twenty hours post transfection, cells were washed with the washing buffer (10 mM HEPES, 142 mM NaCl, 5 mM KCl, 10 mM glucose, pH 7.3) followed by incubation with Chelex-treated DMEM plus 10% FBS. MnCl₂ was added to the media to the final concentration of 50 µM. The other procedures were the same as the Zn transport assay except that the cell lysates were digested with nitric acid and applied to Mn measurement using ICP-MS. More specifically, 75 µL of the cell lysate was mixed with 100 µL of 70% nitric acid (Fisher chemical, Cat# A509P212) in 15 mL metal-free tube (Labcon, Cat# 3134-345-001-9). The samples were heated in 60 °C water bath for 1 h to make sure the sample is fully digested. After incubation each sample was diluted to 3 mL using MilliQ water to a final solution of 2.33% nitric acid (v/v). Samples were analyzed using the Agilent 8900 Triple Quadrupole ICP-MS equipped with the Agilent SPS 4 Autosampler. Instrument calibration was accomplished by preparing standards and internal controls. The standard was IV-ICPMS-71A (Inorganic Ventures), standard concentrations were 100, 50, 25, 12.5, 6.25, 3.125 ng/mL for each element. IV-ICPMS-71D (Inorganic Ventures) was used for internal control. The relative level of Mn was expressed as the molar ratio of Mn and phosphorus^{64 (link)}.

Ultra-pure water (18.2 MΩ∙cm) obtained from a water purification system (Hitech Co., China) was used throughout the experiment. Ultrapure nitric acid (multiple metal element contents below ng/l) and tuning solution (PN#: N8145051) were purchased from Thermo Fisher Scientific Inc. Triton X-100, butanol, 1,4 butanediol, and methanol were all ultrapure and purchased from the Sigma-Aldrich LLC (Shanghai, China). The calibration standards (10 μg/mL), including 43 elements (IV-ICPMS-71A), 12 elements (IV-ICPMS-71B), and internal standards (IV-ICPMS-71D) were all obtained from Inorganic Ventures, Inc. (Christiansburg, USA). Reference reagents for trace elements in sera were obtained from SERO Co. (Trace Element Serum L-1 and L-2, LOT: 1309438, 1309416; Billingstad, Norway).