Human cytokine arrays

Cells were seeded in 10 cm plates (1 × 10⁶ cells/plate) with serum‐free RPMI medium for 24 h. The Conditioned medium (CM) was centrifuged for 20 min at 1000 × g at 4°C to remove particulates and then collected the supernatant to perform the assay. Human cytokine arrays (R&D, SYSTEMS) were incubated overnight with 1 mL of CM at 2−8°C on a rocking platform shaker. After washing with the buffer, the membrane was incubated in diluted Streptavidin HRP in a shaker at room temperature for 30 min. Specific immune complexes were detected using ECL Reagents (Thermo Fisher).

In order to identify soluble factors secreted by senescent and non-senescent cells in culture media, Human Cytokine Arrays (#ARY005; R&D Systems) were used, essentially following the manufacturer’s instructions. Briefly, individual conditioned media (CM) were first combined with the provided cocktail of biotinylated antibodies. The mixture was then incubated with the array membrane. Under these conditions, any cytokine present in the conditioned media would bind to a primary antibody immobilized on the membrane surface. Streptavidin–horseradish peroxidase and chemiluminescence detection reagents (ECL Prime Western Blotting, Amersham, GE healthcare, Chicago, IL, USA) were added, and the signal was detected upon exposure to an X-ray film (Fujifilm super HR-U, Allendale, NJ, USA). Densitometric analyses were then performed. The intensity of the signals was measured using image J software 1.45 (https://imagej.net/Downloads).