Human mouse rat activin a quantikine elisa kit

ELISAs were performed following manufacturers’ instructions using the Human/Mouse/Rat Activin-A Quantikine ELISA Kit (#DAC00B, R&D Systems), the Human IL-6 Quantikine ELISA Kit (#D6050, R&D Systems) and the Human G-CSF Quantikine ELISA Kit (#DCS50, R&D Systems).

Conditional media from the cell clusters isolated from 4‐week‐old control or KC mice were used for mouse cytokines analysis using RayBio Mouse Biotin‐Label Based Antibody Array (Mouse L‐308 Array, Glass Slide). For mouse Activin A quantification, Activin A in conditional media or serum was detected using the Human/Mouse/Rat Activin A Quantikine ELISA Kit (R&D Systems #DAC00B). For human Activin A quantification, Activin A in HPDE conditional media was detected using Human Activin a DuoSet ELISA Kit (R&D Systems #DY338). The amount of Activin A was normalized to the total protein in conditional media or sera and represented as pg mg⁻¹.

Cell culture supernatants were centrifuged at 10,000 × g and 4 ℃ for 10 min. His-tagged ACVR2B-Fc proteins were detected using a His-Tag ELISA Detection Kit (L00436, Genscript). Blood samples of mice were left at room temperature (RT, 20–25 ℃) for 30 min until clot formation. The mouse serum was harvested after centrifugation for 15 min at 2,000 × g. Activin-A and Fc fragment in serum were detected using Human/Mouse/Rat Activin-A Quantikine ELISA Kit (DAC00B, R&D Systems) and Human Fcγ (Fc Fragment of IgG) ELISA Kit (E-EL-H1745, Elabscience). The absorbances at 450 nM were read using an EnVision Multilabel Reader (PerkinElmer).

To determine basal levels of circulating myostatin and activin A, blood was collected at the time of sacrifice from untreated 4‐month‐old male and female Wt and oim/oim mice by cardiac puncture and the serum isolated by centrifugation at 14,000 rpm for 15 minutes and stored at −80°C until assayed. Serum levels of myostatin and activin A were quantified using commercially available ELISA kits, the GDF‐8/Myostatin Quantikine ELISA Kit (DGDF80, R&D Systems, Minneapolis, Minnesota) and the Human/Mouse/Rat Activin A Quantikine ELISA Kit (DAC00B, R&D Systems), respectively. Samples and standards were performed in duplicate following the manufacturer's instructions. A standard curve was generated by plotting the absorbance and concentration values of the standards using a four‐parameter logistic curve fit (online data analysis tool, MyAssays Ltd.) according to the manufacturer's instructions.

The serum levels of Activin A, PlGF and sFLT1 were measured using commercial ELISA kits (R&D, Cat # DAC00B, Human/Mouse/Rat Activin A Quantikine ELISA Kit; Cat # DPG00, Human PLGF Quantikine ELISA Kit; Cat # DVR100C, Human VEGFR1/Flt-1 Quantikine ELISA Kit).