Trypsin edta

Transformed cells HeLa, Cos7, NIH3T3, HEK293 and CHO cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Tet-On 293 cells were maintained in the same DMEM/FBS medium supplemented with 200μg/ml G418 (Gibco-BRL). For transient transfection, cells were grown on Lab-Tak chamber slides (Nunc) and were transfected with plasmid DNAs with Fugene 6 (Roche Molecular Biochemicals) according to manufacturer's instruction. Stable transfection was mediated by electroporation. Stable cell lines or clones were selected at ∼500μg/ml of G418 or 400μg/ml of hygromycin B (Calbiochem).
BJ human foreskin fibroblasts (Stemgent, Cambridge, USA) were cultivated in DMEM with high glucose containing 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, and 30 mM HEPES (Life Technologies, Darmstadt, Germany). Cells were kept at 37°C with 5% CO₂ and media was changed every 3 days. Cells were passaged using trypsin/EDTA (0.04%/0.03%, PromoCell, Heidelberg, Germany). BJ fibroblasts at passage 7-9 were used for all experiments. For transfection, ∼1.5 × 10⁵ cells were plated per well of 12-well plate. Following overnight incubation at 37°C, transfections were performed with Lipofectamine 2000 and cell lines were maintained and selected by NeoR as described above.

HaCaT cells were cultured in T‐175 cm² cell culture flasks at 37°C and 5% CO₂ with the cell culture medium Keratinocyte Growth Medium 2 (KGM2; PromoCell) supplemented with Supplement Mix (PromoCell), 0.06 mM CaCl₂ (PromoCell), and 1% penicillin/streptomycin (PAN Biotech, Aidenbach, Germany). For passaging HaCaT cells, the culture medium was discarded, and the cells were detached from the flask with prewarmed trypsin/EDTA (0.05%/0.02%; PromoCell) at 37°C. The trypsin activity was stopped with FBS (Bio&Sell, Feucht, Germany), and detached cells were collected in a tube and washed twice with prewarmed KGM2. After discarding the supernatant, cells were cultured with KGM2 and seeded in 8‐well chambers, with each well containing a concentration of 5 × 10⁵ cells·ml⁻¹ in preparation for the Dsg3 internalization assay.

Donor male Eng^+/+ mice or Eng^+/− littermates aged 8–12 weeks were killed with an overdose of CO₂. Femurs, tibias, and ilia were surgically dissected, and the adhering tissues were completely removed. Both ends of the bones were excised, and bone marrow cells (BMCs) were harvested by flushing with Endothelial Cell Growth Medium2 (EGM-2), supplemented with fetal calf serum (0.02 mL/mL), VEGF (0.5 ng/mL), basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, ascorbic acid, heparin, hydrocortisone, and antibiotics, using a 25-gauge needle (Promocell, Huissen, The Netherlands). The BMCs were gently resuspended with a 25-gauge needle in EGM-2 medium before culturing on 1% gelatine coated petri dishes (Sigma G9391, bovine skin) at 5% CO₂ at 37°C. Adherent cells were gently washed with PBS at day 3 to remove unattached cells and fresh EGM-2 was added. This procedure was repeated every 2 days until day 14, at which time the BM-derived EPCs were identified by typical endothelial cell (EC) morphology. Petri dishes were washed once with PBS and 1 mL trypsin/EDTA (Promocell) was added. The released cells were counted in a CASY Model TT system (Roche, Almere, The Netherlands) and then re suspended at 10⁶ cells in 100 μL of PBS for transplantation.