Aristar

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).

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.

CRC tissues were lysed with RIPA buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 25 mM nicotinamide, 10 mM sodium butyrate, 1% protease inhibitor cocktail, 1% phosphatase inhibitor A and 1% phosphatase inhibitor B) and homogenized with a gentleMACS Dissociator. The crude lysates were then sonicated on ice for 5 min (25% power, 3 s on and 10 s off), and the protein concentration was determined by Bradford assay (Bio-Rad, 5000205). Then, 400 μl of supernatant was transferred to a new Eppendorf tube and freeze-dried. The lyophilized samples were added to 50 μl of nitric acid (HNO₃) (65% Suprapur, Merck) and digested overnight at room temperature. After heating (90 °C) for 20 min, an equivalent volume of hydrogen peroxide (H₂O₂) (30% Aristar, BDH) was added to each sample. Reheat for 15 min at 70 °C. Then, the samples were diluted with 1% HNO₃ to 1 ml. Measurements were performed using an Agilent 7700 series ICP-MS instrument under routine multi-element operating conditions using a helium reaction gas cell.