Icap qc icp mass spectrometer

Cells cultured in 6-well plates were treated with either BCS or 10–50 μM copper as described above. The plates were then washed 3 times with PBS containing 1 mM EDTA (to remove non-specifically bound copper) followed by the addition of 215 μL concentrated nitric acid (BDH Aristar Ultra). The plates were sealed with parafilm and incubated overnight. After poking a single hole into the caps of 1.5 mL tubes (Sarstedt) using an 18-gauge needle, the digested contents of each well were transferred into the tubes and the samples were boiled at 95 °C for 1 h. Samples (150 μL) were further diluted in 2 mL 2% nitric acid (made freshly from concentrated nitric acid and Milli-Q water) and analyzed on a Thermo Fisher iCAP Qc ICP mass spectrometer in Kinetic Energy Discrimination (KED) mode against a calibration curve of known copper and phosphorus concentrations, with Ga (20 μg/L, Inorganic Ventures) as an internal standard. Each experiment was carried out twice and each condition was repeated in at least triplicate.

Cells were plated in 6-well plates and cultured for one day. The vehicle or compounds were added to the cells the following day and incubated for specified times. NSC319726, 8HQ and MB were added for 24 h. Cobalt chloride was added either alone or in combination with NSC319726 for 24 h. The plates were then washed 2 times with PBS containing 1 mM EDTA and 2 times with PBS alone. After the addition of 215 μL concentrated nitric acid (BDH Aristar Ultra), the plates were sealed with parafilm and incubated overnight. The samples (150 μL) were then diluted in 2 mL 2% nitric acid (ultrapure nitric acid diluted in MilliQ water) and analyzed on a Thermo Fisher iCAP Qc ICP mass spectrometer in Kinetic Energy Discrimination (KED) mode against a calibration curve of known copper, zinc, iron, cobalt and phosphorus concentrations, with gallium (20 μg/L, Inorganic Ventures) as an internal standard. Each experiment was carried out thrice and each condition was repeated in at least triplicate.