To measure circulating irisin levels, Fndc5−/− and Fndc5+/+ mice (n = 5-10 per group) were decapitated and 0.25 mL of trunk blood was collected into EDTA vacutainer tubes to avoid coagulation. Then 100 µL of aprotinin/mL of blood was added to inhibit the protease activity, and tubes were gently shaken and centrifuged at 1600g for 15 minutes at 4 °C. Samples were stored at −20 °C until the assays were processed. A competitive enzyme immunoassay (Phoenix Pharmaceuticals Cat# EK-067-52, RRID:AB_2783013) was used to measure irisin concentration, using manufacturer’s protocol. The assay sensitivity reported by the manufacturer was 1.3 ng/mL, and the intra-assay coefficient of variation was <10%.
Ek 067 52
Manufactured by Phoenix Pharmaceuticals
Sourced in United States
The EK-067-52 is a high-precision laboratory centrifuge designed for a variety of research and diagnostic applications. It features a compact design, quiet operation, and advanced temperature control capabilities to ensure consistent and reliable results. The centrifuge can accommodate a range of sample sizes and tube types, making it a versatile tool for various laboratory workflows.
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4 protocols using ek 067 52
1
Measuring Circulating Irisin Levels
2
Exercise-Induced Biomarker Kinetics
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The subjects presented at 8:00. First, a venous blood sample (i.e., pre-exercise sample) was collected after 30 min of rest. The subjects began prescribed exercise at 8:40. Additional blood samples were collected immediately after exercise (9:10), and at 1 h (10:10), 3 h (13:10), and 24 h (09:10) after exercise (Figure 1 ). The collected blood samples were centrifuged for 10 min (3,000 rpm, 4°C), and the serum and plasma samples were stored at −60°C prior to analysis. Blood samples were assessed immediately after collection with automated glucose (Freestyle; Nipro Co., Osaka, Japan) and lactate (Lactate Pro2 LT- 1730; Arkray Inc., Kyoto, Japan) analyzers. Serum cortisol, myoglobin, and CK concentrations were measured at a clinical laboratory (SRL Inc., Tokyo, Japan). Plasma interleukin (IL)-6 and irisin concentrations were determined using enzyme-linked immunosorbent assay (ELISA) kits [HS600B; R&D Systems, Minneapolis, MN, USA (IL-6); EK-067-52 (Lot 605767); Phoenix Pharmaceuticals, Inc., Darmstadt, Germany (irisin)]. All samples for ELISA were assayed in duplicate and the results were averaged. The intra-assay coefficients of variation were: 2.5% for serum cortisol, 2.4% for serum myoglobin, 2.3% for serum CK, 2.5% for plasma IL-6, and 4.3% for plasma irisin.
3
Measuring Leptin and Irisin Concentrations
4
Irisin Concentration Quantification by ELISA
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Irisin concentrations in serum were determined using a competitive, commercially available ELISA kit (Phoenix, EK-067-52). The linear range for this assay is 0.066–1024 ng/ml (intra-assay variation 4–6 %, inter-assay variation 8–10 %). Absorbance was measured using a spectrophotometer at 450 nm wavelength (Thermo Scientific, Multiskan FC).
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