Human pro bdnf duoset elisa kit

Muscle levels of pro-BDNF (pg mg⁻¹ dry muscle) were quantified using the Human Pro-BDNF DuoSet ELISA kit (DY3175) combined with the DuoSet Ancillary Reagent Kit 2 (DY008) from R&D Systems. Assays were run according to the manufacturer’s instructions with 2 modifications. Samples and standards were diluted in sterile PBS with 10% FBS + 0.02% Tween-20, and then incubated in the coated plate overnight (16 h) at 4°C. All muscle samples were diluted to a final concentration of 0.5 µg µL⁻¹ with 50 µg of protein (100 µL) loaded in each well. The assays were performed with samples and standards in duplicate. A representative standard curve had the following concentration (pg mL⁻¹) and optical density at 450 nm in brackets: 0 (0.156), 78 (0.307), 156 (0.414), 313 (0.623), 625 (0.937), 1250 (1.452), 2500 (2.143), and 5000 (2.847). The optical density for 50 µg of muscle protein generally ranged from 0.400 to 0.600 for the corresponding standard curve.

BDNF, proBDNF and IGF-1 ELISA: Serum BDNF, proBDNF and IGF-1 were measured^{40 (link)} using the Human BDNF DuoSet ELISA kit DY248, Human proBDNF DuoSet ELISA kit DY3175 and Human IGF-1 Quantikine ELISA kit DG100 (R&D Systems, Minneapolis, MN, US). The ancillary kit was used for BDNF and proBDNF. For BDNF measurements, serum samples were diluted 75 ×; the results are shown as the mean of four readings for each sample, each reading representing an independent dilution. For proBDNF measurements, serum samples were diluted 20 × and represent the mean of two independent plates. For IGF-1 quantification, serum samples were pre-treated according to the manufacturer’s instructions (R&D Systems); final dilution of the samples was 100 ×. Samples and standards for IGF-1 ELISA were analyzed in duplicate, and the final values were the average of two readings, each reading representing a separate dilution. Absorbances for BDNF, proBDNF and IGF-1 were read at 450 nm, with a reference reading at 540 nm, using a Multiskan GO plate reader and SKANIT 3.2 software (Thermo-Fisher Scientific, Waltham, MA, USA). BDNF, proBDNF and IGF-1 serum levels were expressed in ng/ml.