D hank s

The skin grafts were washed with D-Hanks (Invitrogen) to remove blood cells, then the subcutaneous adipose cells were removed, and the treated grafts were sheared into 1×1 cm ~ 2×1 cm tissue blocks. These tissue blocks were digested with 2 mg/mL neutral proteinase at 4°C for 10–12 h and further digested at 37°C for 3 h, and thereafter the epidermis was separated. The epidermis was sheared into pieces, digested with 0.25% trypsin-EDTA (Invitrogen) at 37°C for 20 min, combined with DMEM/F12 (Invitrogen) containing 10% FBS to terminate digestion, and blown into a single cell suspension. The cell suspension was grinded and filtered with a 200-mesh sieve. Thereafter, the cell suspension was centrifuged at 1000 rpm for 10 min to collect cells. The supernatant was removed, and the cells were washed once with D-Hanks and then suspended with Epilife medium (Invitrogen) into the plastic culture flask (NUNC) and incubated at 37°C for 20 min. The culture solution was replaced, the suspended cells and impurities were removed, and the cells adhering to the bottom of the culture flask were keratinocytes. The keratinocytes were further cultured in a 37°C, 5% CO₂ incubator. When the cell confluency reached 70%-80%, cells were digested with trypsin for culture passage. The second- or third-generation cells were induced to differentiate by adding 1.5 mM CaCl₂ (Sigma) to the culture medium.

CFs were isolated and cultured as described previously. In brief, neonatal mice (1-2 days old, n = 20) were sterilized in 75% alcohol for 1 min after euthanized with CO₂. Pericardium/epicardium and endocardium were discarded after chest opening while the myocardial tissues (auricles and ventricles) were excised and washed in ice-cold D-Hank's (Gibco, USA) solution and then cut into 1 mm³ pieces. Tissues were then digested with 0.08% pancreatic enzyme (Hyclone, USA) and 0.1% collagenase II (Invitrogen, USA) at 37°C for 8 min with oscillation. The supernatant was collected and filtered through a 400 μm nylon mesh filter and then centrifuged at 1000 g for 8 min. Cells harvested from supernatants were then seeded in 25 cm² flask and cultured with DMEM/F12 (Gibco, USA) contained 10% FBS (Hyclone, USA) and 1% Penicillin-Streptomycin (Hyclone, USA) at 37°C with 5% CO₂ in a humid incubator. The medium was exchanged every other day. CFs were identified with antivimentin (Santa Cruz, catalog number sc-7558, 1 : 200) using immunofluorescence.

EPCs were cultured for 24 hours at 37°C in the presence or absence of ox-LDL. Cells were harvested and adjusted to 5 × 10⁴ cells/mL. Cell migration was quantified by a Transwell chemotaxis assay using a Boyden chamber [17 (link)]. Briefly, 2 × 10⁴ cells (150 μL) were plated in the upper chamber. EBM-2 medium containing 10% FBS (100 μL) was added to the lower chamber. The two chambers were separated by a membrane with 8 μm pores (Corning Transwell). After 24 hours, the membranes were washed twice in D-Hank's (Gibco, USA) and were fixed in 4% formaldehyde. After wiping cells off the upper side of the membrane with a cotton swab (Q-tip), the membranes were detached and mounted on glass slides with 0.25% crystal violet. Migrated cells were counted with a microscopy. Each experiment was performed in triplicate, and the number of migrated cells was determined from 4 random 200x fields per membrane.