Measurement of cellular or lysosomal iron levels

Determination of cellular total, ferric and ferrous iron levels were carried out using the Iron assay Kit
(Abcam) according to manufacturer’s instructions. 

• Previously cultured and bafilomycin-treated MEFs
  were washed in prewarmed PBS, scraped into ice-cold PBS and pelleted at 1000xg for 5 min.
• Cell pellets or purified lysosomes were homogenized in assay buffer on ice using a Dounce homogenizer,
  centrifuged at 16000xg for 10 min and the supernatant collected for the iron assay. 
• 25 uL of samples were made up to 100 uL in a 96-well plate with assay buffer, and incubated for 30 min at 37°C with 5
  uL iron reducer (for total iron) or assay buffer (for ferrous iron) along with standards. 100 mL of iron
  probe was added to each reaction, mixed and incubated for a further 1 hr at 37°C in the dark. 
• The absorbance at 593 nm, which corresponds to the iron levels, was determined using a microplate
  reader (Synergy H1, Biotek).
• Iron concentrations were calculated from the standard curve and normalized to the amount of protein for each sample, determined by the Bradford protein assay. The average of technical triplicates was used for each biological sample.

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