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ICP-MS-CAL-4

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The ICP-MS-CAL-4 is a multi-element calibration standard solution for Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis. It contains a mix of certified standard elements at known concentrations in an appropriate matrix.

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9 protocols using ICP-MS-CAL-4

Briefly, homogenate samples were lyophilised and then digested with nitric acid (65% Suprapur, Merck, St. Louis, MO, USA) overnight, followed by heating at 90°C for 20 min using a heat block. Samples were then removed from the heat block and an equivalent volume of hydrogen peroxide (30% Aristar, BDH, Radnor, PA, USA) added to each sample. Once samples had finished digesting, they were heated for a further 15 min at 70°C. Samples were then diluted with 1% nitric acid diluent. 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, New Haven, CT, USA) for a range of elements, and we also utilized a certified internal standard solution containing 200 ppb of Yttrium (Y89) as a control (ICP‐MS‐IS‐MIX1‐1, Accustandard).
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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).
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Inductively coupled plasma mass spectrometry (ICP-MS) was performed as previously reported [23 (link)]. Briefly, samples were lyophilized and nitric acid (65% Suprapur, Merck, USA) was added for 6-h digestion at room temperature. Samples were heated at 90 °C for 20 min followed by the addition of hydrogen peroxide (30% Aristart, BDH; UAE). Samples were left at room temperature for 30 min before heating again for a further 15 min at 70 °C. The average reduced volume was determined, and the samples were further diluted with 1% HNO3 diluent. 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; USA) for a range of elements. Certified internal standard solutions containing 20 ppb of Yttrium (Y89) as an internal control (ICP-MS-IS-MIX1-1, Accustandard, USA) were used.
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ICP-MS analysis of metal levels was performed as reported previously [24] (link). Cell pellets collected for metal analysis by ICP-MS were re-suspended in 50 µL of concentrated nitric acid (Aristar, BDH, Kilsyth, VIC, Australia) and left to digest overnight. Samples were heated for 20 min at 90°C to complete the digestion. The volume of each sample was reduced to approximately 40–50 µL after digestion, then 1 mL of 1% (v/v) nitric acid diluent was added to each cell sample. Measurements were made using an Agilent ICPMS 7700x series ICP-MS instrument under operating conditions suitable for routine multi-element analysis. The instrument was calibrated using 0, 5, 10, 50, 100 and 500 parts per billion (ppb) of certified multi-element ICP-MS standard calibration solutions (ICP-MS-CA12-1, ICP-MS-CAL-3 and ICP-MS-CAL-4, Accustandard) prepared in 1% (v/v) nitric acid for Cu, Fe, Zn and Mn. A certified internal standard solution containing 200 ppb of Yttrium (Y89) via T-piece was used as an internal control (ICP-MS- IS-MIX1-1, Accustandard). Results were expressed as micromole per liter concentrations of metal (µmol/L). The concentrations of Cu and other metals were calculated as µg of metal per mg of protein based on the protein concentration of the aliquot taken from each sample.
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Tissue samples were coded and processed for inductively coupled mass spectroscopy analysis at the Florey Institute of Neuroscience and Mental Health. Tissue samples were lyophilised and then digested with nitric acid (65% Suprapur, Merck) overnight, followed by heating at 90°C for 20 min using a heat block. Samples were then removed from the heat block and an equivalent volume of hydrogen peroxide (30% Aristar, BDH) added to each sample. Once samples had finished digesting, they were heated for a further 15 mins at 70°C. Samples were then diluted with 1% nitric acid diluent. Measurements were made using an Agilent 7700 series ICPMS 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 ICPMS standard calibration solutions (ICP-MS-CAL2-1, ICP-MS-CAL-3 and ICP-MS-CAL-4, Accustandard) for a range of elements, and we also utilised a certified internal standard solution containing 200 ppb of Yttrium (Y89) as a control (ICP-MS-IS-MIX1-1, Accustandard).
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Frozen cell pellets were lysed in unbuffered 0.5% SDS and cleared by ultracentrifugation. Protein concentration in the lysates was measured with BCA and 500ug of each sample was diluted to a final volume of 400uL with additional 0.5% SDS and freeze dried. Aliquots of 400uL 0.5% SDS as blank controls were included as background controls. The samples were assayed by inductively coupled plasma mass spectrometry (ICPMS; Agilent 7700) under routine multi-element operating conditions using a Helium Reaction Gas Cell, according to a previously published method [34 (link)]. Briefly, samples were treated with concentrated Nitric Acid (65%, Suprapur, Merck) and digested for six hours at room temperature, then heated at 90°C for 20 min to complete the digestion. The instrument was calibrated using 0, 5, 10, 50, 100 and 500 ppb of certified multi- element ICPMS standard calibration solutions (ICP-MS-CAL2-1, ICP-MS-CAL-3 and ICP-MS-CAL-4, Accustandard) for a range of elements, and a certified internal standard solution containing 200 ppb of Yttrium (Y89) as an internal control (ICP-MS-IS-MIX1-1, Accustandard). Cellular copper levels are presented as a ratio of cellular magnesium content.
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ICP-MS was performed as previously described [56 (link)]. Briefly, ipsilateral and contralateral cortical brain homogenates were lyophilized and the dry material was digested with 50 µL of Nitric Acid (HNO3, 65% Suprapur, Merck) overnight. The samples were further digested by heating to 90°C for 20 min using a heating block. Samples were removed from the heating block and an equivalent volume of 50 µL of Hydrogen Peroxide (H2O2, 30% Aristar, BDH) was added to each sample. Samples were allowed to stop digesting for ~ 30 min before heating again for 15 min at 70°C. The average reduced volume was determined and the samples further diluted with 1% HNO3. All measurements were performed on an Agilent 7700 Series ICP-MS instrument (Agilent Technologies, CA, USA) under routine multi-element operating conditions using a Helium Reaction Gas Cell. The instrument was calibrated with 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, CT, USA) for a range of elements. A certified internal standard solution containing 200 ppb of Yttrium (Y89) was used as the internal control (ICP-MS-IS-MIX1-1, Accustandard). Metal content (µg/g) was normalised to protein concentration which was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific).
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Sub-confluent cultures of TBCP-1 cells (5 replicates/condition) were treated for 72 h with vehicle alone (DMSO control) or neratinib (300 nM and 500 nM) and the cells pelleted by centrifugation. Fifty microlitres of concentrated nitric acid (65% v/v, Suprapur, Merck) was added to each cell pellet overnight at room temperature. Samples were heated at 90 °C for 20 min, and final volumes made up to 500 μl 1% (v/v) nitric acid. Iron content was measured using an Agilent 7700 series ICP-MS instrument under routine multi-element operating conditions in 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 standard solution containing 200 ppb of yttrium (Y89) was used as an internal control (ICP-MS-IS-MIX1-1, Accustandard). The raw ppb results were normalised to final volume and converted to ng/106 cell of metal using the formula: Final concentration (ng/106 cell) = Raw ppb value (ng/mL) × sample volume (0.5 ml)/number of cells × 106.
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Tissue of known wet weight was homogenized in Tris buffered saline (pH 7.4) containing EDTAfree protease inhibitor cocktail (1:50, Roche, Dee Why, NSW, Australia) and centrifuged 100 000 g at 4 °C for 30 min. The protein concentration of the resultant supernatant was measured using the BCA protein assay (Pierce, Mt Waverley, VIC, Australia). Subsequently, 50µl of tissue supernatant of known protein concentration was lyophilized by freeze-drying and then suspended in 69% nitric acid (ultraclean grade, Aristar, BDH) overnight. Each sample was heated for 20 min at 90 °C, and an equivalent volume of hydrogen peroxide (30% Aristar, BDH) added followed by a further incubation at 70°C for 15 min. The average volume left after this process was determined to be 35 µL to which 1% HNO3 (800 µL) was added to each tube to give a final sample volume of 835 µL.
The metals in each sample were then measured using an Agilent 7700 series ICPMS under routine multi-element operating conditions and a Helium Reaction Gas Cell. The instrument was calibrated using 0, 5, 10, 50, 100 and 500 ppb of certified multi-element ICPMS standard calibration solutions (ICP-MS-CAL2-1, ICP-MS-CAL-3 and ICP-MS-CAL-4, Accustandard) for a range of elements.
Each sample was measured in triplicate with the concentrations determined from the standard curve of each biometal and normalized to protein concentration.
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