Collagenase type 1

Tissue samples were minced to 1–2 mm pieces and enzymatically digested in DMEM/F12 (Gibco, TFS) containing 2% bovine serum albumin (BSA; Serva, Heidelberg, Germany), 5 mg·mL⁻¹ recombinant human insulin (Sigma‐Aldrich), 0.5 mg·mL⁻¹ hydrocortisone (Sigma‐Aldrich), 50 mg·mL⁻¹ gentamicin (Serva), 2 mg·mL⁻¹ collagenase type I (cat. no. LS 004194; Worthington, Lakewood, NJ, USA), 0.6 U·mL⁻¹ dispase II (cat. no. 04942078001; Roche, Basel, Switzerland) and 10 mm Y‐27632 dihydrochloride (ROCK inhibitor, Santa Cruz Biotechnology, Dallas, TX, USA), for 14 h at 37 °C using 10 rpm agitation. Samples were then treated with 15 mg·mL⁻¹ DNase I (cat. no. 04942078001; Roche) for 5 min at 37 °C, washed with PBS, and filtered through a 70 μm strainer. Red blood cells were lysed with ACK buffer (155 mm ammonium chloride, 10 mm potassium bicarbonate, and 100 μm EDTA solution in sterile MQ water) at 37 °C for 5 min, washed with PBS and incubated with CD24 and ROR1 antibodies 30 min at room temperature. After washing once with CSB buffer, cells were stained with 1 μm cisplatin for subsequent dead cell exclusion (Fluidigm) and quenched with CSB buffer. Cells were then fixed with 1.6% paraformaldehyde (TFS) for 15 min at room temperature and stored at −80 °C in 10% glycerol in fetal bovine serum.

Normal primary fibroblasts (Fb) were isolated and cultured from the normal skin of patients who underwent abdominoplasty. Written consent was obtained. Primary fibroblasts (HSFb and KFb) were isolated and cultured from the hypertrophic scars and keloids, respectively, of 15 patients after obtaining informed consent of the patients and approval of the Shanghai ninth hospital’s Ethical Committee. The mean patient age was 32.7 years (range 22–45 years) and there were nine women and six men. Briefly, the harvested tissue samples were washed with phosphate-buffered saline, cut into pieces and digested with 0.3% collagenase type I (Serva,Germany). The digested tissue samples were filtered through a 200-µm mesh filter, and centrifuged at 500 × g for 10 min. The supernatant was discarded, and the pellet was resuspended in DMEM (HyClone, USA) and cultured in DMEM supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO₂ at 37 °C. Primary cells at passages 2–3 were used for the experiments.