Amphotericin

The assessment was performed on radial growth phase-WM35 human melanoma (Wistar Institute, Philadelphia, PA, USA) [20 (link)] cells and a highly metastatic melanoma cell line M1-15 [21 (link)], donated by professor Andras Falus, Genetics Department, University Semmelweis, Budapest. Melanoma cells were cultured in RPMI medium supplemented with 5% fetal calf serum, 50 μg/ml gentamicin and 5 ng/ml amphotericin, all from Biochrom AG (Berlin, Germany), to avoid the medium influence on the cell lines properties, as previously described [22 (link), 23 (link)]. For all experiments, the cells were used within 4 passages, to preserve their original melanoma characteristics [24 (link), 25 (link)]. Cultures were fed twice weekly and incubated in a humid atmosphere at 37°C and 5% CO₂. All experiments were conducted in triplicate in subdued light, as previously described [26 (link)].

Human umbilical vein endothelial cells (HUVEC-ECCAC obtained from Sigma Aldrich, Steinheim am Albuch, Germany) and dermal fibroblasts (BJ CRL-2522^™—purchased from ATCC, VA, USA) were cultured in RPMI and, respectively, Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal calf serum, 50 µg/mL gentamicin and 5 ng/mL amphotericin, all procured from Biochrom AG (Berlin, Germany). Cultures were fed twice weekly and incubated in standard cell culture conditions. For experiments, the concentration of 2% FCS in medium was used.

Commercial human umbilical vein endothelial cells (HUVEC) were bought from the European Collection of Cell Cultures (ECACC, Porton Down, Salisbury, UK) and multiplied in RPMI medium, supplemented with 10% fetal calf serum (FCS), gentamicin 50 μg/ml, and amphotericin 100 μg/ml (Biochrom AG, Berlin, Germany) in a humidified CO₂ incubator at 37°C. Cell cultures in the 23rd to 26th passages were used. The analysis of surface markers was performed by flow cytometry (BD FACS Canto II flow cytometer, Becton Dickinson & Company, Franklin Lakes, NJ, USA) and used different monoclonal antibodies (ICAM-1, CD29, CD34, CD73, CD90, and CD105).
Cells seeded at a density of 10⁴/cm² in cell culture Petri dishes (TPP) were settled for 24 h in medium then exposed for 24 h to either Equisetum arvense L. extract (25 or 2.5 μg/ml), caffeic acid (10 or 1 μg/ml), and cathechin (10 or 1 μg/ml), as positive controls; afterwards, the cells were washed 3 times with PBS and incubated for 24 h either in normotonic (137 mmol/l) or hypertonic conditions (200 mmol/l). Cells were collected by scraping and treated with a lysis buffer containing IGEPAL-Nonidet 1% (Sigma) and 1% protease inhibitor complex (Sigma) in PBS for 1 h, on ice. The protein content was determined by the Bradford method (Bio-Rad, USA). All the experiments were performed in triplicate.