Phenol red

Primary human aortic endothelial cells (HAEC) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Vascular Cell Basal Complete Medium (ATCC, Manassas, VA, USA) containing 2% of heat-inactivated fetal bovine serum (FBS, ATCC, Manassas, VA, USA), 0.5% of streptomycin-penicillin (ATTC, Manassas, VA, USA), 0.5% of Gentamicin/Amphoterin-B (ATCC, Manassas, VA, USA), 0.5% of Phenol Red (ATCC, Manassas, VA, USA) and an Endothelial Cell Growth Kit (ATCC, Manassas, VA, USA) was used for cell maintenance. Medium was replaced every 2–3 days, and cells were trypsinized before reaching confluence with Trypsin-EDTA (0.05%) Phenol Red (Gibco, Gaithersburg, MD, USA).

The B16F10 melanoma cell lines (ATCC CRL-6475) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Waltham, Massachusetts, USA) high glucose with L-glutamine, phenol red (Wako, Japan) medium containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), and 1% penicillin/streptomycin (P/S) (Nacalai Tesque Inc., Kyoto, Japan).
The human melanoma cell line SK-MEL-28 (ATCC HTB-72) and the human epidermoid carcinoma cell line A-431 (ATCC CRL-1555) were expanded in Eagle’s Minimum Essential Medium containing 10% FBS. Cells of the mouse stromal cell line MS5 and of the murine fibroblast cell line NIH/3T3 were cultured in Iscove’s Modified Dulbecco’s Medium with L-glutamine, and phenol red medium containing 10% FBS and 1%P/S. NIH/3T3 cells (ATCC CRL-1658) were maintained in DMEM. Murine embryonic endothelial progenitor cells (i.e., eEPCs (T17b cells) were cultured in DMEM/10%FBS.

Primary human aortic smooth muscle cells (hASMCs) (ATCC PCS-100-012, American Type Culture Collection, ATCC, Manassas, VA, USA) were maintained in a humidified atmosphere of 37 °C, with 5% CO₂ in VSMC basal medium (without glucose and phenol red) (ATCC^® PCS-100-030™) supplemented with recombinant human basic fibroblast growth factor (5 ng/mL), rhInsulin (5 µg/mL), recombinant human epidermal growth factor (5 ng/mL), L-glutamine (10 mM), ascorbic acid (50 µg/mL), fetal bovine serum (5%), gentamicin (10 µg/mL), penicillin (10 Units/mL), streptomycin (10 µg/mL), amphotericin B (0.28 µg/mL), and phenol red (33 µM) (ATCC). To induce clinically hyperglycemic condition, we stimulated the cells with 25 mM (450 mg/dL) of glucose. Based on our previous study for cell viability [27 (link)], we used 1 and 10 μg/mL concentration of C. turczaninowii extract in this study.

Human Foreskin Fibroblast cells were obtained from the American Type Culture Collection (ATCC, Cat #: SCRC-1041, Manassas, VA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 1 mM sodium pyruvate (Life Technologies), 1x GlutaMAX (Life Technologies), and 1% Penicillin-Streptomycin (Life Technologies). Human Primary Epidermal Melanocyte cells were obtained from ATCC (Cat #: PCS-200-013) and cultured in Dermal Cell Basal Medium (ATCC) supplemented with Phenol Red (ATCC) and Adult Melanocyte Growth Kit (ATCC). Human Melanoma A-375 cells (Cat #: CRL-1619) were obtained from ATCC and cultured in DMEM supplemented with 10% FBS (Life Technologies), 1× GlutaMAX (Life Technologies), 1X Antibiotic-Antimycotic (Life Technologies), and 20 mM HEPES pH 7.4.
Cells were plated on glass-bottom dishes (Willco Wells, Amsterdam, The Netherlands) to < 70% confluence. Cells were left to adhere on the glass bottom dishes overnight and maintained at 37 °C and 5% CO₂. On the following day cells were transported to the AFM system and placed on the AFM X-Y stage to perform force spectroscopy AFM measurements.

Normal human dermal fibroblasts were kept in Fibroblast Basal Medium, supplemented with Fibroblast Growth Kit Serum-Free, Phenol Red and Penicillin-Streptomycin-Amphotericin B Solution (ATCC, Manassas, VA, USA). When appropriate, cells were counted after Hoescht staining (VWR International, Radnor, PA, USA).