Quantichrom iron assay kit

Neutrophil counts were quantified using a Vet-ABC animal blood counter (scil animal care company GmbH, Viernheim, Germany). Determination of serum Tf was performed with an ELISA kit (Abcam) according to the manufacturer’s instructions. Serum iron was measured using the QuantiChrom iron assay kit (BioAssay Systems, Hayward, CA) according to the manufacturer’s instructions. Tf saturation was calculated by using serum Tf and iron concentrations. Analysis of tumor necrosis factor alpha (TNF-α) levels in serum was performed with an ELISA kit according to the manufacturer’s protocol (R&D Systems, Minneapolis, USA). Serum hepcidin was measured with a commercially available kit in exact accordance with the manufacturer’s protocol (Intrinsic LifeSciences).

To determine the possible accumulation of iron in mutant and wild-type colonies of C. minitans, iron concentrations in hyphal masses were measured. Both the mutant ZS-1TN1812 and the wild-type strain ZS-1 were grown on CM-PDA plates for 2 days, and then the mycelial mass was harvested for iron determination. For each sample, the 100 mg mycelial mass was finely ground under liquid nitrogen with a mortar and pestle. The hyphal powder was transferred to a clean 5 mL-tube with 2 mL ice-cold PBS buffer. The mixture was further subjected to sonication with an ultrasonic cleaner (SB5200DT, Wuhan, China) for 20 min, on ice-water. Then, the liquid in the 5 mL-tube was centrifuged at 12,000 rpm for 10 min at 4°C. The clear supernatant was transferred into a clean 2 mL-tube. The supernatant samples were either stored at −80°C or subjected to iron concentration determination. The Quantichrom™ Iron Assay Kit (Bioassay Systems, USA) was used to determine the iron concentration, following the manufacturer's instructions. The iron concentration was measured at a wavelength of 590 nm with an automatic micro plate reader (BOX 998, BioTek® Instruments, Inc. USA). PBS was used instead of a sample as the blank control. This experiment was performed 3 times.

Tissue non-heme iron concentration in renal cortex, outer medulla and liver was measured by a microplate colorimetric assay following acid extraction and reaction with bathophenanthroline reagent as described by Grundy et al.^{42 (link)} Ferric citrate (Avantor^TM Performance Materials, Center Valley, PA) was used to make iron standards for the assay. Iron concentrations were normalized to wet tissue weight. Plasma total iron was measured using the Quantichrom™ Iron Assay Kit (BioAssay Systems, Hayward, CA). BUN and plasma creatinine were measured using Quantichrom^TM urea and creatinine assays, respectively (BioAssay Systems). Commercially available ELISAs were used to measure urine concentrations of transferrin (ADI, San Antonio, TX), ferritin (abcam, Cambridge, UK; also used for plasma) and albumin (Exocell, Philadelphia, PA). Plasma autoantibodies were measured using a mouse anti-dsDNA IgG-specific ELISA kit (ADI). Hemoglobin concentrations were measured in lysed red blood cells and in plasma using Cayman Chemical’s Hemoglobin Colorimetric Assay Kit (Ann Arbor, MI).

Hematological parameters were measured by an automated hematology analyzer KX-21NV (Sysmex Corporation, Hyogo, Japan). The levels of cytokines and albumin present in serum were determined by using commercially available ELISA kits for human IL-6, mouse erythropoietin (EPO) (R&D Systems, Minneapolis, MN, USA), mouse serum amyloid A (Life Technologies Japan, Tokyo, Japan), mouse albumin (Shibayagi, Gunma, Japan), and ferritin (ALPCO Diagnostics, Salem, NH, USA). Serum iron level was determined by QuantiChrom Iron Assay Kit (BioAssay Systems, Hayward, CA, USA). Mouse interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 were measured by Bio-Plex Pro cytokine assays according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA). The assays were performed using the Bio-Plex Pro II wash station with magnetic plate carrier, and cytokines were determined by the Bio-Plex 200 System (Bio-Rad Laboratories).

The Δgrx3Δgrx4 and Δgrx5 yeast strains transformed with either the empty vector, pFL61-RiGRX4, pFL61-RiGRX5, pFL61-RiGRX6 or their respective positive control construct were grown in 50 ml of SD media overnight at 30°C. Each yeast culture was washed twice, resuspended in YPD medium, diluted into 300 ml of YPD medium (OD₆₀₀ 0.2–0.3) and grown until an OD₆₀₀ of 1.0 was reached. Cells were collected by centrifugation, washed with 50 mM Tris pH 7.5, resuspended in 50 mM Tris pH 7.5 containing 0.15 M NaCl and disrupted with glass beads for 10 min at 4°C. Cell homogenates were centrifuged (7000 rpm, 4°C, 5 min) and the supernatants used as cell extracts.
The intracellular iron content was examined with a QuantiChrom^™ iron assay kit (BioAssay Systems, Hayward, CA) following the manufacturer′s instructions.