The unified-ferene assay (u-ferene assay): the finalized protocol

A unified-ferene (u-ferene) assay was developed to enable a 2-in-1 quantification of both labile iron and total iron in a wide variety of biological samples. In this assay, labile and total iron were defined by their experimental parameters; labile iron was defined as iron detected in undigested biological samples using 10 mM ascorbic acid in the working solution, and total iron was defined as iron detected in nitric acid digested biological samples using 1 M ascorbic acid in the working solution.

This assay has five components, (1) working solution, (2) ammonium acetate buffer (pH 4.5, 2.5 M), (3) iron standards, (4) the sample of interest (either cell lysates or biological mice samples), and (5) absorbance measurements.  “Labile iron measurements using the u-ferene assay” outlines the measurement of labile iron and “Total iron measurements using the u-ferene assay”. outlines the measurement of total iron.

The working solution is composed of ferene (5 mM) and ascorbic acid (either 10 mM for labile iron measurements, or 1 M for total iron measurements) in an ammonium acetate buffer. This is summarized in the Supplementary Table ³. The volume of working solution is determined by a 1:6 molar ratio between iron in the highest standard to ferene at 5 mM in working solution—i.e. when using 100 μL samples, working solution is calculated such that there is 6 times more moles of ferene than the moles of iron present in 100 μL of 1000 μM iron standard. Hence, this assay is applicable for investigations requiring larger sample volumes.

Ammonium acetate buffer was added at a 1:1 volume equivalence to all biological samples and iron standards, i.e. when using 100 μL samples, 100 μL of buffer was added.

When using iron standards for u-ferene assay, 1000 μM was used at the highest iron standard, however, this was not considered due to SpectraMax 190 microplate reader’s limit of detection. Iron standards were prepared using FeCl₃ because this is a primary standard used in ICP-MS. The iron standard curve was compared to another primary source, FeSO₄, as well to further validate the labile iron measurements and total iron measurements using the u-ferene assay (Supplementary Fig. ^S5).

Different samples are used. Samples of interest can vary in specimen and complexity, including cell culture lysates to homogenates from mouse tissue samples. It is important to highlight that for labile iron measurements, samples do not require nitric acid digestion, whereas total iron measurements require this digestion to release iron from all stores and proteins.

Details are given in “Absorbance measurements”.

Labile iron concentrations were determined in cell lysates and plasma samples. Samples (either cell lysates or plasma samples) (100 μL) and iron standards (100 μL) with concentrations ranging from 0 to 1000 μM were transferred into different clean Eppendorf tubes. Ammonium acetate buffer (pH 4.5, 2.5 M) (100 μL) and labile iron working solution (5 mM ferene and 10 mM ascorbic acid prepared in ammonium acetate buffer pH 4.5, 2.5 M) (120 μL) were added to all Eppendorf tubes. This mixture was vortexed and left overnight at room temperature. Absorbance was measured as described earlier in “Absorbance measurements”.

Total iron concentrations were determined in cell lysates, plasma samples, organ homogenates, blood, urine, and feces. The nitric acid digested samples after their final dilution (200 μL) and iron standards (200 μL) ranging from 0 to 1000 μM were transferred into different clean Eppendorf tubes. 200 μL of ammonium acetate buffer (pH 4.5, 2.5 M) and 240 μL of total iron working solution (5 mM ferene and 1 M ascorbic acid prepared in ammonium acetate buffer pH 4.5, 2.5 M) were added to all Eppendorf tubes. This mixture was vortexed and left overnight at room temperature. Absorbance was measured as described in “Absorbance measurements”.