Phosphate buffered saline (pbs)

The experimental procedures followed the rules approved by the Ethics Committee of the Luigi Vanvitelli Campania University (n. 0029471/i). Patients were informed of the research and gave permission for the use of biological samples. Bone marrow aspirate samples were obtained from eight healthy donors (age 10–18 years). We separated cells on a Ficoll (GE Healthcare) density gradient and, after centrifugation at 400g for 30 min, the mononuclear cell fraction was collected. We seeded 1–2.5 × 10⁵ cells/cm² in complete medium composed by alpha‐MEM (Microgem) containing 10% FBS (EuroClone), 4 mM l‐glutamine (EuroClone), 100 U/ml penicillin–streptomycin (HiMedia) and 3 ng/ml FGF2 (Peprotech). The cells were then incubated at 37°C in a humidified atmosphere containing 5% CO₂. After 24 h the non‐adherent cells were discarded and the adherent cells at passage 0 (P0) were washed twice in PBS (Microgem) and re‐incubated in complete medium for 7–10 days until maximum confluence was reached. We used the minimal criteria suggested by the International Society for Cellular Therapy⁷ to identify Mesenchymal Stromal Cells (MSCs). The MSCs have been expanded up to passage 4 (P4). Subsequently, the cells were used to isolate MUSE cells and an aliquot was frozen in FBS with 10% DMSO (Sigma) in liquid nitrogen.

The experimental procedures followed the rules approved by the Ethics Committee of the Luigi Vanvitelli Campania University. Patients were informed of the research and gave permission for the use of biological samples. Bone marrow was obtained from three healthy donors. Cells were separated through Ficoll density gradient (GE Healthcare, Chicago, IL, USA), and the mononuclear cell fraction was collected and washed in phosphate-buffered saline solution (PBS, Microgem, Naples, Italy). We seeded 1 to 2.5 × 10⁵cells/cm² in alpha-minimum essential medium (alpha-MEM, Microgem) supplemented with 10% fetal bovine serum (FBS, Euroclone, Pero, Italy) and 1 ng/mL beta-fibroblast growth factor (β-FGF, Prepotech, London, UK). After 72 h, non-adherent cells were discarded, and adherent cells were further cultivated to reach confluency. We verified that, under our experimental conditions, the bone-marrow stromal cultures contained MSCs that fulfilled the three criteria proposed to define MSCs [35 (link)]. All experiments were conducted on MSC cultures at passage 3 when senescence phenomena were minimal [36 (link)].

Following UV exposure, cells were fixed in a solution containing 2% formaldehyde. Subsequently, the cells underwent thorough washing with phosphate-buffered saline (PBS) (Microgem, Italy). They were then incubated at a temperature of 37°C for an overnight period within a staining solution. This solution consisted of citric acid/phosphate buffer (pH 6), K₄Fe (CN)₆, K₃Fe (CN)₆, NaCl, MgCl₂, and X-Gal. To assess the proportion of senescent cells, a count was conducted on the cells displaying β-galactosidase-positive characteristics, indicated by their blue coloration. This count was performed across multiple microscopic fields, encompassing no fewer than 300 cells in each instance, adhering to the methodology described in previous work [38 (link)]. Unless specifically mentioned, all reagents utilized in this study were procured from Sigma-Aldrich (MO, USA).

Twenty thousand MUSE cells per well were seeded in 24 wells with glass coverslips. Cells were fixed in a solution of 2% formaldehyde for 10 min. Then, cells were washed with PBS (Microgem) and incubated at 37°C overnight with a staining solution (citric acid/phosphate buffer (pH 6), K₄Fe (CN) 6, K₃Fe (CN) 6, NaCl, MgCl₂) containing 1 mg/ml of X‐Gal (GoldBio).¹⁰ The percentage of senescent cells was calculated by the number of cells that expressed the marker stain out of at least 500 cells in different microscope fields.