Cells were grown on 100-mm tissue culture dishes and analyzed for metals by inductively coupled plasma mass spectrometry (ICP-MS) as we described previously (Choi et al. 2020 (link); Choi et al. 2019 (link); Choi et al. 2018 (link)). Briefly, the cell samples were digested with 2 mL/g total wet weight nitric acid (BDH ARISTAR®ULTRA) for 24 h and then digested with 1 mL/g total wet weight hydrogen peroxide (BDH Aristar® ULTRA) for 24 h at room temperature. The samples were stored at 4°C until metals were quantified. Ultrapure water was used for final sample dilution. For mitochondrial iron levels, mitochondria were isolated through differential centrifugation as we previously described (Choi et al. 2018 (link)), and then mitochondrial iron levels were measured via ICP-MS.
Aristar ultra
Manufactured by BD
Sourced in United States
ARISTAR®ULTRA is a high-purity laboratory reagent designed for trace analysis. It is produced using a specialized purification process to ensure minimal impurities, allowing for reliable and accurate results in sensitive analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
1.5 ml tube, by Sarstedt (1 mentions)
Cms 5, by Inorganic Ventures (1 mentions)
Icap qc icp mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Chloroform, by Thermo Fisher Scientific (1 mentions)
Nuclease free water, by Qiagen (1 mentions)
6 protocols using aristar ultra
1
Quantification of Cellular Metal Levels
2
Measuring Cellular and Mitochondrial Metals
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Cells were grown on 100‐mm tissue culture dishes and analyzed for metals by inductively coupled plasma mass spectrometry (ICP‐MS) as we described previously (Choi et al., 2018 (link), 2019 (link), 2020 (link)). Briefly, the cell samples were digested with 2 ml/g total wet weight nitric acid (BDH ARISTAR®ULTRA) for 24 h and then digested with 1 ml/g total wet weight hydrogen peroxide (BDH Aristar® ULTRA) for 24 h at room temperature. The samples were stored at 4°C until metals were quantified. Ultrapure water was used for final sample dilution. For mitochondrial iron levels, mitochondria were isolated through differential centrifugation as we previously described (Choi et al., 2018 (link)), and then mitochondrial iron levels were measured via ICP‐MS.
3
Quantitative Analysis of Cellular Elemental Composition
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Cells were then rinsed twice with ice-cold potassium free buffer (150 mM Na+ in 50 mM HEPES buffer, pH = 7.4), followed by the addition of 215 μL of concentrated nitric acid (BDH Aristar Ultra). The plates were sealed with Parafilm and incubated on a shaker overnight. Samples (150 μL) were further diluted in 2 mL of 2% nitric acid (made freshly from concentrated nitric acid and Milli-Q water) in 15 mL tubes (Sarstedt) and analyzed on a Thermo Fisher iCAP Qc ICP mass spectrometer in kinetic energy discrimination (KED) mode against a standard curve of known potassium and phosphorus concentrations (CMS-5, Inorganic Ventures), with Ga (20 μg L−1, Inorganic Ventures) as an internal standard. Each experiment was carried out twice, and each condition was repeated in at least triplicate.
4
Quantifying Metals in hSLC39A8-Expressing Cells
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HeLa cells transfected with hSLC39A8-WT or other mutants were analyzed for metals by inductively coupled plasma mass spectrometry (ICP-MS) (Lumigen Instrument Center, Department of Chemistry, Wayne State University, MI, USA), as described previously38 (link),41 (link). Briefly, the cell samples were digested with 2 mL/g total wet weight nitric acid (BDH ARISTAR®ULTRA) for 24 h, and then digested with 1 mL/g total wet weight hydrogen peroxide (BDH Aristar® ULTRA) for 24 h at room temperature. The samples were preserved at 4 °C until quantification of metals. Ultrapure water was used for final sample dilution.
5
Quantitative Metal Analysis in Cells
6
Rigorous Polymerware Preparation for Trace Analysis
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All experiments were performed in polymerware (polypropylene or polycarbonate), which was first soaked in 2% v/v HNO3 for 24 hours, rinsed 7x with Milli-Q water (total organic carbon < 2 µg L -1 ; resistivity > 18 MΩ cm) and dried under laminar flow conditions. Chemicals were molecular biology grade or higher, including acetic acid (analytical grade, Fisher Scientific, CA); chloroform (99,8%, Acros organics, CA); nuclease-free water (Qiagen, USA); K2HPO4 and KH2PO4 (ACS reagent grade, Fisher Chemical, CA); Tris (Tris-(hydroxymethyl)-aminomethane, USP/EPgrade, BDH, CA); EDTA disodium salt (Bioultra grade, Sigma-Aldrich, CA); Isotone (VWR, CA); HNO3 (67-70%; Aristar Ultra, BDH), NaOH (Acros Organics, CA), NaMES (2-(N-morpholino)ethanesulfonic sodium salt, Acros Organics, CA); NaHEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic sodium salt, Acros Organics, CA). Single element (1.0 g.L -1 ; Ce(NO3)3, Tm(NO3)3, Y(NO3)3) and multielement (10 mg.L -1 ; CMS-1) ICP-MS standards were acquired from Inorganic Ventures (USA).
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