Senescence detection kit

AT biopsies (3–5-mm thickness) were fixed for 1 h with a fixative solution provided by the Senescence detection kit (Biovision Inc., Milpitas, CA, USA). After washing with PBS, samples were stained with the staining solution (X-Gal substrate: 20 mg/ml), provided by the Senescence detection kit (Biovision), and incubated at 37°C (without CO₂) for 20–22 h. After, AT samples were first fixed in 4% paraformaldehyde overnight at 4 °C and then embedded in paraffin blocks.
From each adipose specimen, serial paraffin sections (4 μm thick) were obtained and counterstained with Fast Red (Sigma-Aldrich, Milano, Italy). Tissue sections were examined with a Nikon Eclipse E800 light microscope and digital images were captured with a Nikon camera (DXM 1200). Senescence-associated (SA)-β-galactosidase (Gal) positive (+) cells and adipocytes were counted by optical microscopy. The number of senescent cells was expressed as percentage in relation to adipocytes by counting the number of SA-β-Gal + cells and the number of adipocytes in at least 20 fields. The analysis excluded cells within and around blood vessels.

For the assessment of cellular senescence, nHHDPCs (2×10⁶) were seeded into 60-mm cell culture dishes and treated with 0 or 400 μM PPD. Following 48 h of treatment, cells were fixed using Fixative solution (included in Senescence Detection kit; BioVision, Inc., Milpitas, CA, USA) and senescence-associated-β-galactosidase (SA-β-gal) activity was measured using the Staining Solution Mix, including Staining Solution, Staining Supplements and X-gal substrate for (SA-β-gal) within the Senescence Detection kit, according to the manufacturer’s instructions. Cells stained for SA-β-gal were counted under a light microscope (CKX41; Olympus Corporation, Tokyo, USA) (magnification, ×200) and the percentage of SA-β-gal positive cells were calculated.

β-Galactosidase (SA-β-GAL) activity was detected using the senescence detection kit (BioVision, Milpitas, CA, United States). Cells were fixed with a fixable solution (senescence detection kit, Bio Vision Milpitas) for 15 min at room temperature (approx. 25°C). Fixed cells were then washed with PBS (−) and treated with staining solution, staining supplements, and X-gal solution (final concentration: 1 mg/mL; BioVision, Milpitas, CA, United States), and incubated overnight at 37°C.