2.6. Luminex assays

Conditioned media (CM) was collected by culturing cells or islets in growth media lacking FBS for 18–24 h. CM was collected, clarified by centrifugation (3000 g for 5 min at room temperature) and either used immediately or stored at 4 °C for up to 2–3 weeks. Luminex assays were carried out on CM from cells treated as indicated using a custom magnetic bead kit that included mouse SASP factors IL-6, Serpine1 and Igfbp3, or human SASP factors CXCL1, CXCL8/IL-8, IGFBP4, GDF-15, or TNFRSF10C (R&D systems). 50 μl of CM was assayed on a Bio-Plex 200 luminex platform (Bio-Rad) as previously [7,14] and secreted amounts in 1–2 ml of CM were normalized to viable cell counts to calculate amount secreted per 100,000 cells or for human islets secretion was normalized to total RNA content. Alternatively, media was spin-concentrated using a 3 kDa MWCO centrifugal unit (Millipore-Sigma) and the concentrated conditioned media was analyzed by Luminex.