Iron standard for aas

Oven dried liver samples weighing 4–9 mg were added to 300 μL of concentrated nitric acid. Duplicate samples for each liver were then digested in a heated sand bath. Chromogen reagent was prepared (0.1% banthophenanthroline sulfate, 1% mercaptoacetic acid). One part of chromogen reagent was added to five parts of saturated sodium acetate to make working chromogen reagent immediately prior to use. When fully digested, the volume of each sample was determined by pipette and 25 μL was added to 125 μL working chromogen reagent. Absorbance was measured at 540 nm on a plate reader, Tecan infinite F200, Tecan, Switzerland. The iron concentration by dry weight was determined with reference to an iron standard (Iron standard for AAS, Sigma, St. Louis).

MΦ were stimulated either with LPS (0.5 μg/ml/IFNγ (100 U/ml) or IL-10 (20 ng/ml) for 24 h. Where indicated, IL-10 stimulation was supplemented with the chelators (TC3-S)₂ at a concentration of 100 μM for the last 4 h of incubation. The iron content of MΦ supernatants was determined by graphite furnace atomic absorption spectrometry. Samples were measured as triplicates with a PinAAcle^™ 900 T Atomic Absorption Spectrometer (PerkinElmer, Waltham, USA). Slit 0.2 nm and wavelength 248,33 nm were used as spectrometer parameters. A hollow cathode iron lamp (30 mA maximum operating current) was run at 100% maximum current. The calibration solutions (10 μg/l to 90 μg/l) were prepared by adequate dilution of Iron Standard for AAS (Sigma-Aldrich, Steinheim, Germany) stock solution. A pyrolysis temperature of 1400°C and an atomization temperature of 2100°C were used.