Asx 500 autosampler

Trace elemental analysis was conducted using an Agilent 8800 ICP-MS (Agilent Technologies, Santa Clara, CA, USA). The sample introduction system for the ICP-MS included a MicroMist nebulizer (Glass Expansion, Pocasset, MA), a Scott double-channel spray chamber (2 °C), and a shield torch. The ICP-MS was used in collision mode with helium gas at 3.0 mL/min to reduce isobaric interferences. Isotopes monitored included ⁵⁵Mn, ⁵⁶Fe, ⁵⁹Co, ⁶³Cu, and ⁶⁶Zn. For total metal analysis, an Agilent ASX-500 Autosampler was employed and ⁴⁵Sc was used as internal standards. For cell lysate separation, an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA) with a Zorbax GF-250 column (Agilent Technologies, Santa Clara, CA, USA) was used. The HPLC was equipped with a vacuum membrane degasser system, binary pump, cooled autosampler, a temperature-controlled column compartment (held at 25 °C), and a diode array detector where the UV absorbance was recorded at 280 nm. The integrated area of the Gel Filtration Standard was used to normalize the ICP-MS signal from day to day. The HPLC was coupled to the ICP-MS allowing for nearly simultaneous UV detection and metal detection. Total protein concentration was determined using a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).