Elisas

Two-month-old WT and Ocn^–/– mice born from Ocn^+/+, Ocn^+/–, or Ocn^–/– mothers received vehicle (0.3% BSA in PBS) or recombinant ACTH (A0298, MilliporeSigma, 1 μg/g) via i.p., injection at 1000 hours. Injections were done sequentially between 1000 hours and 1025 hours. Blood samples were obtained between 1030 hours and 1100 hours from facial vein within 10 seconds of handling of the mice to minimize stress-induced changes in adrenal steroid levels. Blood was collected in serum-separating tubes (Microvette 500 Z Gel, Starstedt), allowed to clot for 30 minutes, and centrifuged at 13,523g for 10 minutes at 4°C to obtain serum. Only 2 drops of blood were collected from each mouse, and serum volumes varied between 10 μL and 20 μL per mouse. Circulating corticosterone and aldosterone levels were determined by ELISAs (Abcam) in duplicate according to the manufacturer’s instructions. Serum (2 μL) was used for corticosterone and 5 μL for aldosterone measurements with biological replicates. Internal controls from a pooled serum sample collected from adult mice were used in each assay to calculate inter- and intra-assay variations. The interassay variation was less than 7%–10%, and the intra-assay variation was less than 1%–2%.

After the treatment was finished, 100 μl peripheral blood was collected from each group and congealed at room temperature for 20 minutes. The supernatants were harvested by centrifugation at 3000 r.p.m. for 5 minutes. The concentrations of IL-6 and progranulin (PGRN) were measured by ELISAs (Abcam).

ELISAs (Abcam) were used to detect the levels of ANGPTL4 and VEGF and were performed according to the manufacturer’s instructions. The concentration of each sample was determined by measuring the absorbance at 450 nm in a microplate reader. Each sample was run in triplicate.

Pi was measured using a colorimetric kit (Abcam), and iFGF23 and PTH were measured by the respective Immutopics ELISAs (catalog nos. 60-6800 and 60-2305) according to the manufacturer’s instructions. Colorimetric kits for calcium (Stanbio) and blood urea nitrogen (Invitrogen) were used, and 1,25(OH)₂D was measured by enzymatic immunoassay (IDS), while creatinine was measured by liquid chromatography–mass spectrometry (LC-MS) as previously described (10 (link)).

Blood samples were collected within 24 h after diagnosis, during the worst period of disease, and 2–3 days prior to death or discharge (blood samples were collected from the patients every two to three days to monitor for all biochemical markers in our center). The serum levels of FGF21, interleukin 6 (IL-6), IL-10, tumor necrosis factor α (TNFα), procalcitonin (PCT), and C reactive protein (CRP) were determined using enzyme-linked immunosorbent assays (ELISAs) from Abcam (USA) according to the manufacturer’s instructions. Routine blood tests and blood gas analyses were performed in the hospital’s central laboratory.