4.2. Cytokine Antibody Arrays

Cytokine antibody arrays were performed on human follicular fluid using the Ray Bio C-series human cytokine antibody array C5 (Ray Biotech Inc, Norcross, GA, USA) according to the manufacturer’s instructions. Human follicular fluid samples from each participant were run in duplicate, and 500 μL of follicular fluid was used on each array. Although media contamination in the follicular fluid samples was anticipated to be low because only the first follicle from each ovary was used, we performed cytokine antibody arrays on media alone to determine the background and, thus, establish signal thresholds. The cytokine antibody arrays were visualized using chemiluminescence according to the manufacturer’s instructions. The cytokine antibody arrays were imaged, and the Protein Array Analyzer Plugin for ImageJ was used to quantify signal intensity using densitometry [68]. The region of interest for each cytokine measured was kept constant across all experiments. Intensity values were exported into Ray Biotech’s analysis tool, and intensity values were normalized to the oldest participant. Relative intensity values for each cytokine were plotted versus age, BMI, and AMH values.