Hepes

Plasma heme was measured using 1-Step™ Turbo 3,3',5,5'-Tetramethylbenzidine (TMB) enzyme-linked immunosorbent assay (ELISA) Substrate (Thermofisher scientific). This method is based on the oxidation of TMB by the pseudoperoxidase activity of heme. For this assay, hemin chloride porcine (designated as heme, Sigma-Aldrich) was diluted in 0.1 M NaOH (Merck) and the pH was adjusted to 7.8–8.2 (29 (link)). Subsequently, this solution was filtered through 0.2 µm pore size membrane (Whatman). Hereafter, serial dilutions (4.0–2.0–1.5–1–0.5–0.25–0 µM) of a 2 mM heme stock were made in 20 mM HEPES (Sigma-Aldrich) + 1% bovine serum albumin (BSA, Millipore), pH 7.4. Next, plasma samples (patients 10×, controls 2.5×) were diluted in 20 mM HEPES + 1% BSA, pH 7.4, where after 20 µl of every sample and 80 µl of the turbo TMB substrate was added to a microtiter plate (Greiner Bio-One™) and incubated for 10 min in the dark. The reaction was stopped with 100 µl 2 M sulfuric acid (Sigma-Aldrich) and absorbance was read at 450 nanometer (nm) with Victor 3 V multilabel plate reader (PerkinElmer).

All cell culture reagents were purchased from Sigma-Aldrich (Dorset, England) unless otherwise specified. Pancreatic insulinoma RINm5F β-cells (ATCC, CRL11605, Rockville, MD, USA) from Rattus norvegicus were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (32404, Gibco, Loughborough, England) supplemented with 10% (v/v) fetal bovine serum (F9665), 1 mM sodium pyruvate (11360070, Gibco, Loughborough, England), 4500 mg/L glucose (G8644), 2 mM Glutamax supplement (35050061, Gibco, Loughborough, England), 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and maintained at 37 °C, 5% CO₂, and 95% relative humidity in tissue culture flasks (Greiner Bio-One, Kremsmünster, Austria). Cultures were routinely split (1:2) at ~80% confluency by rinsing the cells with Dulbecco’s phosphate buffered saline (DPBS, 10 mm) without calcium or magnesium and released for sub-cultivation using 0.25% (v/v) trypsin-EDTA.