Magpix calibration and performance verification kits

Blood was collected from females by cardiac exsanguination under ketamine/xylazine anesthesia, and collected serum was stored at −20 °C. Mice were collected at the diestrus stage for cycling mice and pseudo-diestrus for acyclic PCOS mice. FSH and LH levels in 10 µl of sera diluted in 15 µl serum dilution buffer (1 in 2.5 dilution) was measured using EMD Millipore’s MILLIPLEX® MAP 96-well Mouse Pituitary Magnetic Bead custom Kit (Cat# MPTMAG-49K) according to manufacturer’s instructions (EMD Millipore, Billerica, MA, USA). Luminex MAGPIX instrument (Luminex Corporation, Northbrook, IL, USA) calibrated with MAGPIX Calibration and Performance Verification Kits (Millipore) with xPONENT software (Luminex) was used to acquire data. Saved data was then analyzed as the Median Fluorescent Intensity (MFI) using spline curve-fitting for calculating analyte concentrations in samples with Multiplex Analyst software Version 5.1 (Luminex).

A Cytokine and Chemokine 26-Plex Mouse ProcartaPlex^TM Panel 1 (Thermofischer Scientific, Waltham, MA, USA) was used to assess cytokine levels in mouse plasma samples, according to the manufacturer’s instructions. We used a Luminex MAGPIX instrument (Luminex Corporation, Northbrook, IL, USA) calibrated with MAGPIX Calibration and Performance Verification Kits (Millipore, Burlington, MA, USA) with xPONENT software (Luminex) to obtain data. Data were analysed using Multiplex Analyst software version 5.1 (Merck, Darmstadt, Germany) as the Median Fluorescent Intensity (MFI) using spline curve-fitting for calculating analyte concentrations in samples.