Element 2 icp ms

Freeze-dried and homogenised tissue (~0.1 g) was digested in HNO₃ (5 mL, 68%) in pressurised closed vessels (Mars 5, CEM Corporation, UK). The microwave programme was as follows: step 1, room temperature to 200 °C, 20 min; step 2, 200 °C, 30 min. After the completion of the microwave programme, the digests were allowed to cool to room temperature and were stored at 4 °C until elemental analysis. Sample analysis was performed by ELEMENT 2 ICP-MS (Thermo Fisher Scientific, Germany). Prior to measurement, the digests were diluted in bi-distilled water to a final acid concentration of 5% and m/z ⁷⁷Se, ⁸²Se and ⁷⁴Ge were monitored during the measurement in high-resolution mode.

AX2 cells were grown for 24 h in M1 ± Fe medium or for 480 h in M2 ± Zn or M3 ± Cu media. A total of 10⁸ cells were washed five times in Soerensen phosphate buffer and pelleted in Eppendorf tubes. Digestion was performed by adding 1 ml of concentrated HNO₃ (70%) to each sample. After complete dissolution, samples were further digested by applying microwave heating (Milestone MicroSYNTH, Microwave labstation equipped with an optical fiber temperature control and HPR-1000/6M six position high-pressure reactor, Bergamo, Italy). After digestion, the volume of each sample was brought to 3 ml with ultrapure water, filtered with 0.45 mm filter and analyzed by ICP-MS, using a Thermo Scientific ELEMENT 2 ICP-MS (Finnigan, Rodano, Italy) (Fiorito et al., 2012 (link)). The same analysis was also performed on 800 μl of M2 or M3 medium. The quantification was obtained through a calibration curve measured by using six Fe/Cu/Zn absorption standard solutions (Sigma-Aldrich) in the range 0.0025–0.3 μg/ml. Samples with metals concentrations higher than the upper limit were diluted opportunely. Sample digestion and metal quantifications were carried out by the facility of the Molecular Imaging Center (Department of Chemistry, University of Torino, Italy).

At the end of the MRI experiments, the animals were sacrificed by cervical dislocation, in agreement with ethical rules. Cancer tissues, kidneys, liver, spleen, and muscles were explanted, weighed, and processed for ICP-MS analysis [53 (link)]. The tissues were added to 1 mL of concentrated HNO₃ (70%). Upon dissolution of the tissues, samples were further digested through microwave heating (MicroSYNTH, Microwave labstation, Microsynth, Balgach, Switzerland, equipped with an optical fiber temperature control and HPR-1000/6M high-pressure reactor, Milestone, Bergamo, Italy). After digestion, ultrapure water was added to each sample up to a 2 mL total volume, then samples were filtered with 0.45 μm filter and analyzed using ICP-MS for quantification of Gd³⁺, using a Thermo Scientific ELEMENT 2 ICP-MS-Finnigan, Rodano, Italy. A calibration curve obtained by using four gadolinium absorption standard solutions (Sigma-Aldrich, Burlington, MA, USA) in the range 0.005–0.1 μg/mL was used for the quantification. The total mass of Gd³⁺ retained in each specimen was calculated with respect to the weight of the tumor tissue (as μg of Gd³⁺/g of tissue).

The Lanthanide content of cells was determined using ICP-MS. Cells were collected in 0.1 mL PBS and destroyed by sonication before being digested with concentrated HNO₃ (70%) under microwave heating (MicroSYNTH). After mineralization, each sample was taken with 2 mL of ultrapure water and analyzed using an ELEMENT 2 ICP-MS (Thermo Scientific). Three replicates of each sample solution were analyzed.

CDDP was administered i.v. at 5:00 or 17:00 every 7 days for 4 weeks (n = 6). Blood samples were obtained from the tail vein at 5, 15, 30, 60, 120, 240, 360, 480, and 720 min after the first and fourth administration of CDDP. All blood samples were centrifuged immediately at 3000 × g for 10 min at 15 °C. Lumber 4 (L4), L5, and L6 DRG samples were isolated from each anesthetized rats at 24 h after the first and fourth administration of CDDP. Serum and DRG samples were frozen at −80 °C until analyzed.
In order to determine serum CDDP concentrations, 10 μL of rat serum was mixed with 990 μL of 60 % HNO₃, with Indium ICP-MS standard (Wako Pure chemical Industries, Ltd.) as an internal standard. Each DRG was mixed with 2000 μL of 60 % HNO₃, with Indium ICP-MS standard as an internal standard, to determine total CDDP concentrations in DRG. This solution was incubated at 100 °C for 30 min, and then diluted 100 times with ultrapure water.
Total platinum concentrations were assayed using inductively coupled plasma mass spectrometry on an ELEMENT2™ ICP-MS (Thermo Fisher Scientific, Inc.). The operating parameters of the ICP-MS instrument were as follows: RF power 1280 W, cool gas flow 15.58 L/min, auxiliary gas flow 0.80 L/min, sample gas flow 0.98 L/min, dead time 25 ns, take-up time 1 min, and four replicates per sample.