Collagenase f

Implantation sites were harvested as described above. Each site was cut into 12 pieces and placed in RPMI 5% fetal bovine serum (FBS). These pieces were digested in RPMI containing 5% FBS, 300 μg/mL collagenase F (Sigma‐Aldrich), 200 μg/mL collagenase L (Sigma‐Aldrich), 500 μg/mL Dispase (Gibco), and 2 U/mL DNase‐1 (Roche) at 37°C for 30‐45 minutes with a magnetic stir bar for agitation. Cells were passed over a 70 μm strainer (BD) to create a single‐cell suspension. Implantation sites were washed in DPBS, 1% FBS, 25 mmol/L EDTA. Cells were stained for FACS with anti‐CD45 (30‐F11, BD), anti‐CD11b (M1/70, BD), anti‐Gr‐1 (RB6‐8C5, BD), and rabbit anti‐Crry followed by a donkey anti‐rabbit DyLight 488 (Jackson ImmunoResearch). Blocking for FACS was carried out employing DPBS, 1% FBS, 25 mmol/L EDTA with 5% donkey serum, and 5% mouse serum. Cells were examined employing a FACScan (BD) retrofitted with a Cytek Upgrade.

Mouse external ear pinnae were harvested and separated into dorsal and ventral leaflets. To ensure standardized sampling and equal sample sizes, ears lobes were excised just above the cartilage area of the outer ear. For Fig. 2A, a 2 cm² piece of challenged skin from the back torso was excised. Subcutaneous tissue was scraped off and the skin was finely minced and digested with 0.25% collagenase F (Sigma-Aldrich, Dorset, UK) for 30 minutes at 37°C. Digested tissue pieces were then mashed through a 70-μm sieve in order to generate a single cell suspension. The cell mixture was directly used for flow cytometric analysis of skin infiltrating cells or further processed for isolation of CD45⁺ or CD4⁺ cells by FACS sorting. For in vitro IGF-1 stimulation experiments, Foxp3EGFP mice were used and the CD4 population was FACS-sorted into CD4⁺GFP⁺ (total CD4) and CD4⁺GFP⁻ (Treg cell-depleted CD4) populations. FACS sorting was performed using FACS Aria or FACS Aria SORP (Becton Dickinson, Oxford, UK, 70 μm nozzle, 70 psi; >98% purity).

For isolation of cells from heart, lymph nodes and spleen, mice were perfused in situ with ice-cold PBS through the apex of the left ventricle to clear blood from the circulation. To generate single-cell suspensions, tissues were excised, finely minced using surgical scissors on ice and digested with 0.25% collagenase F (Sigma-Aldrich) in RPMI medium for 30 min at 37°C with gentle agitation as described (Johannesson et al., 2014 (link)). After the first round of digestion, supernatants were filtered through 70 µm filters and transferred into 10% FBS and 300 U ml⁻¹ DNaseI (Roche Diagnostics, Burgess Hill, UK) on ice to block enzyme activity and washed twice with ice-cold PBS for 5 min at 1200 rpm, 4°C. Undigested tissue pieces were transferred into fresh collagenase solution for a second round of digestion for 30 min at 37°C.
For in vitro culture experiments, excised spleens were gently mashed through a 70 μm cell strainer, washed and subjected to red blood cell lysis using Red Blood Cell Lysis Buffer (Sigma-Aldrich) according to the manufacturer's instructions. The obtained cell mixture was then used for flow cytometric analysis.

The rat VSMCs were derived from male Wistar rats. Briefly, male Wistar rats aged 10 weeks with a weight of 200 ± 20 g were euthanized by cervical dislocation. Mesenteric arteries were dissected gently from the abdominal aorta, the fat and connective tissues were carefully separated from the arteries, and the arteries were cut into 1 mm long cylindrical sections. These sections were incubated in solution containing 1.5 mg/mL Papain, 1.5 mg/mL dithiothreitol, and 1.5 mg/mL BSA for 20–25 min at 37°C, followed by digestion in 1.0 mg/mL collagenase F, 1.5 mg/mL hyaluronidase, and 1.5 mg/mL BSA for 6-10 min. Papain, dithiothreitol, BSA, collagenase F, and hyaluronidase were purchased from Sigma-Aldrich (St. Louis, MO, USA). After digestion, the single smooth muscle cells were dissociated by gentle trituration with a pipette. Cells were maintained in DMEM with the supplementation of 20% fetal bovine serum (Gibco, USA) and 100 U/mL streptomycin/penicillin (Gibco, USA).
The protocol was approved by the Animal Care and Welfare Committee of the First Affiliated Hospital of Harbin Medical University. The experimental protocols were conducted in accordance with the National Guidelines for the Care and Use of Laboratory Animals.