4.5. Cytokine Release Experiments

Female-derived or male-derived hRMCs described above were serum-starved for three hours in serum-free complete MCM (without FBS supplementation) when ~80% confluent. Human sera were then added to a final concentration of 5%, incubated for three hours, and refed with fresh serum-free complete MCM. Serum from a single individual was used for all experiments. All treatments were performed in duplicate or triplicate. Media was collected from hRMCs following incubation with human sera. Cell viability was then measured using the Alamar Blue assay (Invitrogen/ThermoFisher, Waltham, MA, USA) following the manufacturer’s instructions. ELISA kits from Biolegend (San Diego, CA, USA) were used to measure CCL5 (RANTES) or CXCL5 according to the manufacturer protocol. Relative cell viability (per well) with respect to untreated cells was used to normalize measured cytokine levels per well. For the individual serum analyses, replicates for each serum donor were averaged. The averages for each serum donor are shown on the graphs as individual points.