Procedures

Procedure 1: Administration of diets varying in iron content, and plasma and liver harvest

Fasting (duration): 4h before tissue harvest

Equipment, software, and supplies

• Heparin-coated capillary tubes
• Lithium-heparin collection tubes (BD Biosciences, 365965)

Reagents and solutions

• Low-iron diet, 12 ppm iron (Dyets, 115111)
• Sufficient-iron diet (Control), 50 ppm iron (Dyets, 515005)
• High-iron diet, 20,000 ppm iron (Dyets, 115122)
• Isoflurane
• PBS
• Liquid nitrogen

Steps

1. At four weeks of age, mice are fed either a low-iron diet, high-iron diet, or sufficient-iron diet (control). Mice are fed their respective diets for 6 weeks.
2. Mice are fasted for 4h at 10 wks of age.
3. Blood is taken from the retro-orbital plexus under isoflurane anesthesia using a heparin-coated capillary tube; blood is placed in a lithium heparin collection tube for plasma preparation.
4. Mice are euthanized and then perfused via the heart with PBS to flush remaining blood from the tissues. Livers are harvested and frozen in liquid nitrogen and stored at -80°C until analysis.

Procedure 2: Hepcidin ELISA

Fasting (duration): 4h

Equipment, software, and supplies

• High-binding 96-well enzyme immunoassay plates (Corning Costar, 3590, Tewksbury, MA, USA)
• 96-well plate reader (Molecular Devices, Sunnyvale, CA, USA)

Reagents and solutions

• Monoclonal antibody to mouse hepcidin-1 (capture) (Amgen, Ab2B10, Thousand Oaks, CA, USA)
• Horseradish peroxidase (detection) (Amgen, Ab2H4-HRP, Thousand Oaks, CA, USA)
• Synthetic mouse hepcidin-25 (Amgen, Thousand Oaks, CA, USA)
• Carbonate-bicarbonate buffer, pH 9.4 (Pierce-Thermo Scientific, Rockford, IL, USA)
• PBS and 0.5% Tween 20 (wash buffer)
• PBS, 1% BSA, 1% normal goat serum, 0.5% Tween 20 (blocking buffer)
• Ultra-TMB substrate (Thermo Scientific)
• Sulfuric acid

Steps

1. High-binding 96-well enzyme immunoassay plates are coated overnight at room temperature with 50 µL/well of 3.6 µg/mL Ab2B10 in 0.2 M carbonate-bicarbonate buffer, pH 9.4.
2. Plates are washed with wash buffer and then incubated 45 min in blocking buffer.
3. Plasma samples are diluted between 250 and 10,000 times in blocking buffer.
4. Standards are placed in wells in duplicate. A standard curve is generated by diluting the stock mouse hepcidin peptide (50 ng/µL) to a final concentration of 200 pg/mL for the highest standard followed by two-fold dilutions in blocking buffer.
5. After a 1h incubation at room temperature with mixing, wells are washed and then incubated with 50 µL/well of 130 ng/mL Ab2H4-HRP.
6. Wells are developed with 100 µL/well Ultra-TMB substrate for 15-30 min in the dark at room temperature.
7. The reaction is stopped by by addition of 50 µL of 2 M sulfuric acid.
8. Absorbance is measured at 450 nm using a plate reader.

Procedure 3: Liver RNA purification and qRT-PCR for hepcidin

Equipment, software, and supplies

• Tissue homogenizer (Pro Scientific, Bio-Gen Pro-200, Oxford, CT, USA)
• Centrifuge

Reagents and solutions

• TRIzol (Invitrogen, Grand Island, NY, USA)
• Chloroform
• Isopropanol
• Nuclease-free water
• cDNA kit (Bio-Rad, iScript, Hercules, CA, USA)
• SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA)
• Primers for hepcidin and beta-actin

Steps

1. Total RNA is extracted from frozen mouse livers using the TRIzol method.
   1. TRIzol (1 mL) is added to a small piece of tissue (~50 mg) and homogenized.
   2. Chloroform is added and shaken vigorously and then centrifuged at 13,000 g to separate organic and aqueous phases.
   3. RNA in the aqueous phase is precipitated using isopropanol and then dissolved in nuclease-free water.
2. cDNA is made from total RNA according to manufacturer's instructions using 250 ng of total RNA with the following conditions: 25°C for 5 min, 42°C for 30 min, 85°C for 5 min.
3. qRT-PCR reactions are run according to manufacturer's instructions with a 2-step program set at 95°C for 10 s, 60°C for 30 s for 35 cycles. Beta-actin is used to normalize the data. See McLachlan et al., 2017 for primer sequences.