D4263

In the chronic study, lung tissue and BAL cells were prepared for flow cytometry analysis. Lung tissue was incubated with collagenase (1 mg/ml, 234153, Clostridium histolyticum, Calbiochem, Germany) and DNase (2 U/ml, D4263, Sigma-Aldrich, USA) in PBS, after which a single cell suspension was prepared. Cells were then centrifuged for 5 min at 1,500 rpm at 4°C. Red blood cells were removed using shock buffer (containing 8.3 g/l NH₄Cl, 1 g/l KHCO₃ and 37 mg/l EDTA). Cells were washed and centrifuged for 5 min at 1,500 rpm at 4°C. The pellet was resuspended and plated in a 96 wells plate (Nunc MaxiSorp®, eBioscience) after which the cells were stained with the live/dead staining Aqua (Life technologies, Bleiswijk, The Netherlands). After fixing the cells in a 1.9% PFA solution for 15 min at RT, they were washed and resuspended in FACS buffer, after which they were stained for flow cytometric analysis. Antibodies that were used are listed in Table 4. Mouse FcγRII/III-binding inhibitor (kindly provided by Louis Boon, Bioceros) was added to the antibody mix. Measurements were performed on a FACSCanto II (BD Bioscience, San Jose, CA) and analyzed using FlowJo (v7.6.5) software (Tree Star, Ashland, OR).

After euthanasia, the skin of irradiated mice was shredded using a sharp blade. Subsequently, the skin was incubated in DMEM containing 0.5 mg/mL collagenase I (A004194, Diamond), 0.5 mg/mL collagenase II (A004174, Diamond), 1 mg/mL collagenase IV (A004186, Diamond), 0.5 mg/mL hyaluronidase (H3506, Biosharp), 0.5 mg/mL elastase (A002290, Diamond) and 12.5 kU DNase (D4263, Sigma). Incubation was performed at 37 °C for 90 min. After incubation, cells were filtered through a 70 μm cell strainer. Subsequently, cells were treated with FcR blocking reagent (156604, Biolegend) for 10 min at 4 °C, then stained with fluorescently labeled CD26 antibody, and dead cells were filtered using DAPI. Senescent cellular fibroblasts with DAPI⁻ CD26⁺ tdTomato⁺ and non-senescent fibroblasts with DAPI⁻ CD26⁺ tdTomato⁻ were isolated using a BD FACS Aria II, and culture was continued in DMEM.

Tissue samples were digested as follows: 10 mg pieces of murine postischemic hearts were digested with Collagenase II (1 mg mL⁻¹; C6885, Sigma) and DNase I (50 µg mL⁻¹; D4263, Sigma) for 1 h. Porcine biceps femoris samples were sectioned into 50 mg pieces, digested first with 1 mg mL⁻¹ papain (P3375, Sigma) for 30 min, followed by 1 mg mL⁻¹ Collagenase/Dispase (Roche) for 30 min. Cell suspensions were pressed through a cell strainer (70 µm) and centrifuged at 300 × g. Red blood cells were lysed with an ammonium‐potassium buffer washed twice and stained for viability with trypan blue for murine and LIVE/DEAD (Thermo Fisher) for porcine cells. Murine cells were immediately stained with a specific antibody for CD31 (BM4086 , Origene) for 15 min, followed by secondary antibody staining (A48265, Thermo Fisher). Porcine cells were fixed with 4% w/v paraformaldehyde, permeabilized with Triton X100 and stained for EGFP (CAB4211, Thermo Fisher) and CD31 (LCI‐4, Bio‐Rad), followed by secondary antibody staining (A32731 and A48255, Thermo Fisher). Fluorescence measurements were carried out in an LSRFortessa (BD Biosciences).