Agilent 7700

Total metal concentration was measured using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700, Varian). The cells were washed with 5 mL PBS before harvesting with 2 mL of trypsin-EDTA solution. The suspended cells were counted. The cell suspension was centrifuged at 800 rpm for 5 min at room temperature to pellet the cells and subsequently stored at -80 °C until further use. The cells or Lf samples were prepared for ICP-MS analyses as previously described 39 (link). Triple determinations of iron concentration were performed for each sample. Unit conversions from raw ppm values were performed as follows: raw ppm values × dilution factor/cell number.

For Se analysis culture samples (50 mL) were centrifuged and sediments of cells were freeze-dried before analysis. Biomass was mineralized in the microwave decomposition equipment (Milestone 1200 Mega) using nitric acid and hydrogen peroxide. The total amount of Se in biomass was determined using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700×) by a commercial company (Healthcare Institute Ltd., Ústí nad Labem, Czech Republic).

Al³⁺ treatment was given in the form of aluminium chloride (AlCl₃) prepared in 0.5 mM CaCl₂ (pH 4.5). After treatment, the hairy roots were washed three times with deionized water. The hairy roots were cut and collected for Al measurement, respectively. Next, 0.5 g of tissue sample was placed in a tank containing 5 ml of HNO₃ and 2 ml of H₂O₂ for 4 h and was digested in a microwave, followed by dilution to 25 g with water as the sample solution. The Al content was measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Agilent 7700, USA).

The mineral levels in the feed, feces, liver, spleen, and kidney were measured by inductively coupled plasma optical emission spectrometry (ICP-OES) (Agilent 7700, Agilent, Santa Clara, CA, USA) as described previously (1 (link), 4 (link)).

The oven-tried samples were ground to a fine powder through a ball mill (MM 200, Retsch GmbH, Haan, Germany) and digested (0.5–0.7 g) with 10 mL of concentrated HNO₃ (65%) at 190 °C for 45 min in a microwave digester (Mars 6, CEM Corporation, Matthews, NC, USA) following protocols described by [19 (link)]. The digests were diluted to 50 mL with double DI water and stored for lateral analysis. The concentrations of Se, Cd, Zn, Cu, and Mn were determined through inductively coupled plasma mass spectroscopy (ICP-MS; Agilent 7700, Agilent Technologies Inc., Böblingen, Germany), and concentrations of Ca, Mg, P, S, Fe, and Na were determined through Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) as described by Wu et al. [24 (link)]. For making standard curves, a multi-element ICP-standard solution (ROTI^®STAR, Roth, Germany) was used. The Cd uptake and translocation characteristics were calculated using the following Formulas (1)–(4);

The determination of Cd in different components (cell wall (CW), soluble fraction (SF), and organelle fractions (OF) was carried out following the methods described by Wu et al. [19 (link)] with some modifications. Briefly, fresh samples of the root, young leaves, and old leaves (1 g) were ground with extraction solution (0.25 M sucrose, 50 mM Tris-HCl buffer (pH 7.5), and 1 mM dithioerythritol) in chill conditions. The homogenate was then filtered, and the obtained pallet was rewashed with the extractant. For cell wall extraction, the filtrate was centrifuged (300× g for 30 s), and the obtained pallet, together with the washed pallet, was considered a cell wall component. The filtrate obtained was centrifuged again at 20,000× g for 45 min using a Micro-centrifuge (Thermo Scientific™ Heraeus, Hanau, Germany), and the obtained pallet (OF) and filtrate (SF). For Cd determination, the soluble fraction was diluted and used directly, whereas SW and OF components were wet digested with 10 mL HNO₃ (65%) following laboratory protocol using a microwave oven digestion system (Mars 6, CEM Corporation, Matthews, NC, USA) and diluted before Cd was determined through inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7700, Agilent Technologies Inc., Böblingen, Germany).