Antibiotic antimycotic solution

This assay was performed essentially as described previously [6 (link)] with minor modifications. HT-29 cells obtained from the ATCC^® (Manassas, VA. Lot number 4487730) were derived from colon epithelial cells. Cells (10⁵ cells per 50-ml flask) were grown in 7 ml DMEM supplemented with 10% FCS, 1% glutamine, 1% antibiotic-antimycotic solution (Biological Industries, Bet Haemek, Israel) and in the presence or absence of either 1 μM trT2-50, mock (equivalent amount in PBS) or control (PBS). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and the growth medium was changed 24 h after cell implantation. After another 24 h, cells were seeded in 96-well plates (1 × 10³ cells/well/200 μl medium) in the presence or absence of 1 μM trT2-50, mock (equivalent amount in PBS) or PBS. After 5 days, the cells were fixed in 5% formaldehyde, 60% ethanol, and 5% acetic acid in water and stained with methylene blue. In each treatment, colonies that contained at least 100 cells were counted. Six individual determinations were performed for each treatment.

RPMI 1640 medium, fetal bovine serum (FBS), antibiotic-antimycotic solution, L-glutamine, and trypsin/EDTA were purchased from Biological Industries, Beit HaEmek, Israel. Rho 123, DMSO and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma–Aldrich Chemie GmbH, Hamburg, Germany. FITC-conjugated anti-P-gp antibody was purchased from BD Biosciences, Plymouth, UK. A Pgp-Glo^TM assay system was obtained from Promega, Madison, WI, USA.

Dulbecco’s Modified Eagle’s Medium (DMEM), sera and antibiotic–antimycotic solution were purchased from Biological Industries (Beit-Haemek, Israel). Sirius red F3B was obtained from BDH Laboratory Supplies (Poole, UK). The LDH kit CyQUANT was purchased from Invitrogen (Eugene, OR, USA).

Freshly isolated HUVEC were maintained in endothelial cell growth medium (EGM) supplemented with SingleQuots (EGM BulletKit CC-3124, Lonza). They were then plated in a 96-well plate (14 × 10³ cells/well) previously coated with growth factor-depleted Matrigel (Becton-Dickinson, Bedford, MA) in M199 medium supplemented with 20% FCS, 1% glutamine, 1% antibiotic–antimycotic solution (Biological Industries) and 50 U/100 ml heparin (Biomedical Technologies Inc., Stoughton, MA). Cells were simultaneously treated with either trT2-50 (2 μM) or PBS, in addition to angiogenin (R&D Systems, Inc. Minneapolis, MN), or VEGF (Protein Laboratories, Rehovot, Israel) (1 μg/ml each). After 8 h of incubation, at 37°C, the plates were photographed, and the extent of tube formation was counted using Image J (NIH, Bethesda, MD) software. Five individual determinations were performed for each treatment.

The in vitro biocompatibility testing of the materials was performed on two commercial cell lines, the L929 (mouse fibroblasts) and RAW 264.7 (mouse macrophages) cell lines, both obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The L929 cell line was taken for indirect culture system testing on fibroblasts, as it is the most commonly used model for testing dental materials on fibroblasts. The RAW 264.7 cell line is a macrophage line used for phagocytosis assay and immunomodulatory activity testing of materials. The cells of both cell lines were cultured in Dulbecco’s Modified Eagle Medium with 1 g⁄L glucose (DMEM) supplemented with 2 mM stable glutamine, 1% antibiotic-antimycotic solution, and 10% fetal bovine serum (complete DMEM), all purchased from Biological Industries (BioInd, Kibbutz Beit-Haemek, Israel), in standard cell culture conditions (humidified atmosphere, 5% CO₂, 37 °C).