Icp ms cal2 1

Soil samples were collected using a 9-point sampling method, with these points distributed in a Z-shape in each plot, each point being collected at a distance of 50–70 cm away from the vine in the row, at three depths: 0–30 cm, 30–60 cm, and 60–90 cm. Soils collected at the same depth from the nine sites were completely mixed and then divided into three replicates for analysis of particle content, organic matter, electrical conductivity, pH, and cation exchange capacity (CEC). Soil from the 30–60 cm depth, which is the root enrichment zone, was used for analysis of soil mineral elements. The determination of basic soil physico-chemical properties was based on Han [17 (link)]: Soil pH was measured in KCl solution with a soil/solution ratio of 1:2.5 v:v; organic matter was determined via sulfochromic oxidation; electrical conductivity (EC) was measured with a conductivity meter; and CEC was determined via the ammonium acetate method [18 (link)]. N was determined via the Kjeldahl method, which consists of three steps: sample digestion, distillation, and ammonia determination [19 (link)]. P, Fe, Ca, K, and Mg contents were determined via inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7800, Santa Clara, CA, USA). First, 0.25 g of soil was taken, and 5 mL of HNO₃ was added for digestion. Secondly, the solution was heated at 100 °C for 30 min before cooling. After cooling and heating until nearly dry, 1 mL of H₂O₂ was added. After cooling, the double-distilled water volume was reduced to 50 mL and analyzed. The ICP-MS was equipped with an autosampler, a Burgener nebulizer, nickel cones, and a peristaltic sample delivery pump. Detection parameters were as follows: 15 L/min plasma gas flow, 4.3 mL/min helium and reaction gas flow, 0.90 L/min carrier gas flow (>99.99% argon purity), 0.3 r/s sample lift rate, and an atomization chamber temperature at 2 °C. An external standard method was used for quantification, which was prepared with a multi-element standard solution (ICP-MS-CAL2-1, AccuStandard, New Haven, CT, USA) in 0.5% HNO₃ (chromatographically pure) as described by Wu [20 (link)].

50 μL of soluble fractions were lyophilized for 12 h and then digested with 50 μL of nitric acid (65% Suprapur, Merck) overnight (~12 h) at ~22 °C. Further digested the samples by under heating conditions (90 °C for 20 min). Samples were then removed from the heating block and an equivalent volume of 50 µL hydrogen peroxide (H₂O₂) (30% Aristar, BDH) was added to each sample. Samples were allowed to stop effervescing, for 30 minutes, before heating again for a further 15 minutes at 70 °C. Then cooled to ~22 °C. Samples were further diluted with 1% HNO₃ diluent up to 500 μL (dilution factor 1:10) and iron and copper were measured by inductively coupled plasma-mass spectrometry (ICP-MS), using an Agilent Technologies 7700× ICP-MS system (Agilent Technologies, Australia) under routine multi-element operating conditions. As previously described [29 (link)], helium (3 ml/min) was used as the collision gas to minimize polyatomic interferences with all elements. The instrument was calibrated using 0, 5, 10, 50, 100 and 500 ppb of certified multi-element ICP-MS standard calibration solutions (ICP-MS-CAL2-1, ICP-MS-CAL-3 and ICP-MS-CAL-4, Accustandard) for a range of elements. Used a certified internal standard solution containing 200 ppb of Yttrium (Y89) as an internal control. The samples were analysed without identification. For each sample, iron, copper and zinc levels were corrected for protein concentration determined by BCA protein assay (Cat 23225, Thermo Fisher Scientific, USA) and expressed in μmol/g protein.

For measurement of total zinc 5.0 × 10⁵ cells for each cell line (PNT1A, LNCaP, DU145 and PC3) were cultured in 60mm cell culture dishes in 5mls serum media overnight. Next day serum media was aspirated and cells were washed briefly for 10–20 seconds with 2mLs of Milli-Q water. A final volume of 500μL of Milli-Q water was added and cells were scraped and the lysate collected into a 1.5 mL eppendorf tube. Cell lysates were freeze-dried, nitric acid (50 µL of 65%, Suprapur, Merck) was added to each cell pellet, and the pellets were digested overnight at room temperature. The samples were heated using a heating block at 90°C for 20 min to a volume of ∼40 µL. To each sample 460µL of 1% (v/v) of nitric acid diluent was added to a final Volume of 0.5 mL. Measurements were made using an Agilent 7700 series ICP-MS instrument under routine multi-element operating conditions using a helium reaction gas cell. The instrument was calibrated using 0, 5, 10, 50, 100 and 500 ppb of certified multi- element ICP-MS standard calibration solutions (ICP-MS-CAL2-1, ICP-MS-CAL-3 and ICP-MS-CAL-4, Accustandard) for a range of elements. A certified internal standard solution containing 200 ppb of Yttrium (Y89) was used as an internal control (ICP- MS-IS-MIX1-1, Accustandard).

Detergent lysed SLMV’s, from equal numbers of fractionated cells, were digested in concentrated high purity nitric acid (Aristar; BDH, London, UK) overnight at room temperature, and then at 90 °C for 20 minutes. Samples were diluted with 1% nitric acid, and measurements made using a Varian UltraMass inductively coupled plasma mass spectroscopy (ICPMS) instrument (PaloAlto, CA ,USA) under operating conditions suitable for routine multi-element analysis. The instrument was calibrated using blank, 10, 50, and 100 ppb of a certified multi-element ICPMS standard solution (ICP-MS-CAl2-1; AccuStandard) for Mn²⁺, Fe²⁺, Cu²⁺, and Zn²⁺ in 1% nitric acid. Results were obtained from four independent experiments. Mn²⁺ and Fe²⁺ were below levels of detection and Cu²⁺ values were inconsistent across the experimental groups. Zn²⁺ concentrations were untransfected < transfected mutant < transfected wild-type in all experiments. Zn²⁺ in un-transfected cells was deducted from the values in transfected cells prior to analysis. Comparisons between wild-type and mutant were made within experiments, with the wild-type value set as 1.