Mouse high sensitivity t cell magnetic bead panel

Serum was collected by centrifugation at 1000 g for 15 min at 4°C, aliquoted, and stored at −80°C until analysis. A Mouse High Sensitivity T Cell Magnetic Bead Panel (EMD Millipore, Billerica, MA, Unites States) was performed on the Luminex-MAGPIX multiplex immunoassay system according to the manufacturer’s instructions. The data were analyzed using Milliplex Analyst 5.1 software (EMD Millipore, Billerica, MA, United States).

After 8 weeks of treatment, blood samples were obtained from the mice by cardiac puncture at the time of sacrifice. The samples were centrifuged at 800× g for 15 min at 4 °C, and the serum samples obtained were stored at −80 °C until subsequent analysis. The serum concentrations of IL-6, IL-1β, and TNF-α were measured using a Mouse High-sensitivity T Cell Magnetic Bead Panel (Merck Millipore, Burlington, MA, USA). The serum concentrations of estradiol and IGF-1 were measured using a Mouse Estradiol ELISA Kit (MyBioSource, San Diego, CA, USA) and an IGF-1 ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively. All these analyses were performed according to the manufacturers’ instructions. Absorbances were measured at appropriate wavelengths using a Luminex 100 analyzer (Luminex, Austin, TX, USA). All the sample concentrations were measured in triplicate.

A mouse high-sensitivity T-cell magnetic bead panel was obtained from Merck Millipore (Darmstadt, Germany), and the experiment was conducted according to the manufacturer’s instructions. All samples were measured in duplicate. Standard curves of known concentrations of recombinant human cytokines/chemokines were used to convert fluorescence units to cytokine concentration units (pg/mL). All results below the minimum concentrations were processed as the minimum concentrations.