Trypsin edta 0.5

A549 (ATCC^® CCL-185™), CuFi-1(ATCC^® CRL-4013™) and NuLi-1 (ATCC^® CRL-4011™) cells were bought from the American Type Culture Collection (ATCC; Manassas, VA, USA). Roswell Park Memorial Institute (RPMI) 1640 medium without phenol red, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), penicillin-streptomycin (PEST), inactivated fetal bovine serum (FBS), Dulbecco’s Phosphate Buffered Saline (DPBS) and trypsin-EDTA (0.5%) without phenol red were purchased from Gibco™ (Life Technologies, Madrid, Spain). Serum-free Bronchial Epithelial Growth Medium (BEGM Bullet Kit; CC-3170) made of BEBM basal medium and SingleQuot additives; Airway Epithelial Cell Basal Medium and Bronchial Epithelial Cell Growth Kit additives were purchased from Lonza (Clonetics, Lonza, Walkersville Inc., Walkersville, MD, USA). Dimethyl sulfoxide (DMSO) was purchased form Scharlau (Madrid, Spain). Human Placental Collagen Type IV (Sigma Cat. No. C-7521) and Cell Counting Kit-8 (CCK-8) were bought from Sigma-Aldrich (Saint Louise, MO, USA). OxiSelect™ Cellular Antioxidant Cell Kit was bought from Quimigen (Madrid, Spain) to Cell Biolabs, Inc. (San Diego, CA, USA).

Neuro2a mouse neuroblastoma cells (N2a, Department of Neuroanatomy and Molecular Brain Research, Ruhr-University Bochum, Bochum, Germany) were cultured in T75 flasks in DMEM (DMEM, high glucose, GlutaMAX Supplement, Gibco) with 1% 10,000 units/ml penicillin/streptomycin and 5% FBS. Cells were split using trypsin-EDTA 0.5% (Gibco) at a confluency of 90%. For experiments, cells were detached, heated for 10 min in 90°C PBS and cooled down on ice for another 10 min to secure cell death.

Primary Human Dermal Fibroblast (NDHF Promo Cell #FB60C12350) supplied by Carlo Erba were used as the control cell line and compared with primary dermal fibroblast from a young Marinesco–Sjögren syndrome patient, supplied by Telethon Network of Genetic Biobanks—TNGB [36 (link)]. Cells were cultured in Dulbecco’s modified Eagle’s medium + GlutaMAX (GIBCO, 61965-026), supplemented with 10% of Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin PenStrep (GIBCO, 15070-063). Cells were maintained at 37 °C, 5% CO₂, and detached by Trypsin-EDTA 0.5% (GIBCO, 15400-054).

Resident CMSCs were isolated by primary explant cultures. Whole atria and ventricle samples were mechanically cut into 1–2 mm³ fragments, washed in PBS, partially digested in trypsin-EDTA 0.5% (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) for 3 min, and finally plated as primary explant cultures on fibronectin (FN)-coated dishes (Cell Guidance Systems, Cambridge, UK). Explants were cultured in standard incubators in complete explant medium (CEM)—IMDM (Gibco), 1% penicillin—streptomycin, 1% L-glutamine, 0.1 mM β-mercaptoethanol, and 0.1% Primocin (InvivoGen, San Diego, CA, USA)—supplemented with 20% FBS (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA). To evaluate the outgrowth yield, a scoring system was set up: score 0 = no or few cells within a 200 µm radius; score 1 = confluent cell outgrowth beyond a 200 µm radius (Figure 1a). After 3 weeks, CMSCs were collected as outgrowth explant-derived cells by gentle trypsinization [39 (link)] and frozen. Once thawed, CMSCs were plated on FN-coated dishes and used after one week for all experiments.

To evaluate the dose-response at given concentrations and the time-course of Eos, the cells where detached with Trypsin-EDTA 0.5% (Gibco, USA) and 5 × 10³ cells/well were seeded and incubated in a 96-well cell plate with DMEM-F12 supplemented with 10% FBS for 24 h. The supernatant were removed and the cells were washed 1× with PBS 1X (Gibco, USA), and treated with EOs extracts of CA or LS at known concentrations (64, 32, 16, 8, 4, or 2 µg/mL) dissolved in culture medium (DMEM-F12 supplemented with 1% FBS). The vehicle was DMSO 1% dissolved in DMEM-F12, supplemented with 1% FBS as a control treatment (the EOs are dissolved in DMSO 1%). After 24 or 48 h the supernatant was discarded, the cells were washed with PBS 1X and incubated with 100 µl of crystal violet (CV) solution (0.2% w/v in ethanol 10%) (Gibco, USA) for 20 min, then the CV solution was removed and Na₂HPO₄ (0.1 M, pH 4.5, in ethanol 50:50 v/v) (SIGMA, USA), was added to elute the intra-cellular colorant. The absorbance of each sample was measured at 570 nm. The results are shown as percentage of color intensity and normalized to cells grown in DMSO 1% as control treatment.

Trypsin-EDTA 0.5% (Gibco, USA), PBS 1X (Gibco, USA), DMSO, crystal violet, ethanol, Na₂HPO₄, DMEM F12, FBS, MTT, HCl, were purchased from Sigma-Aldrich (St. Louis, MO, USA).