96 well black plate

An angiogenesis assay plate (BD Pharmingen) was used to assess the angiogenetic ability of EPCs. 96-well black plates (BD Pharmingen) with clear bottom uniformly coated with BD Matrigel Matrix were incubated for 30 minutes at 37°C with 5% carbon dioxide. The EPCs were added at 1 × 10⁵/well at different time points. The samples were incubated for 24 hours at 37°C with 5% carbon dioxide. For each plate, 6.25 mL Hank's balanced saline solution (HBSS) (BD Pharmingen) was mixed with 20 μL dimethyl sulfoxide (BD Pharmingen) and 50 μg Calcein AM (BD Pharmingen). After incubation, the medium was carefully removed from the plates. The plates were washed by adding 100 μL HBSS to each well, and the number of EPC formed tubes was counted by fluorescence microscopy (IX71; Olympus, Tokyo, Japan).

The human colon cancer cells (SW480) were obtained from ATCC (American Type Culture Collection, Manassas, United States) were maintained in DMEM supplemented with 10% FBS, 1% antibiotic–antimycotic mix at 37°C under a humidified 5% CO₂ and 95% air atmosphere. Cells were exposed to 0.25% trypsin-EDTA and harvested cells were seeded at a density of 1 × 10⁴ cells/well on 24 well-plates, 6-well plates (Costar, United States) and 96-well black plates (BD Biosciences, Franklin Lakes, NJ, United States) for different assays.

Human umbilical vein endothelial cells (HUVECs) were obtained from Cell Applications (Cell Applications, San Diego, CA, USA) and cultured as previously described [43 (link)]. HUVECs were sub-cultured at a split ratio of 1:2 and used within three passages before cell viability was checked using trypan blue. Unless not otherwise specified, cells were plated in 96-well black plates (BD Falcon, Franklin Lakes, NJ) and processed for experiments in Medium M199 containing 5% FCS plus the different treatments. RES 10 mM stock solution was prepared in dimethyl sulfoxide (DMSO); final DMSO concentration in the diluted treatment media was less than 0.1%. DMSO (less than 0.1%) was used as a vehicle control. To investigate the involvement of ROS and NADPH oxidase in the RES-induced cellular effects, in selected experiments, cells were pretreated for 1 h with either 100 µM of the broad antioxidant Tempol [50 (link)] or with 10µM of the flavin-oxidase inhibitor Diphenyleneiodonium (DPI) [49 (link),51 (link),52 (link)]. The involvement of PKC in HUVECs response to RES treatment was investigated by using 2.5 µM of the broad PKC inhibitor chelerythrine (CHE) [53 (link),54 (link)].

Cell apoptosis was assessed by using the fluorimetric kit APOPercentage (Biocolor Ltd., Carrickfergus, UK) following the protocol provided by the manufacturer. This assay has been employed with several adherent cell lines including endothelial cells [37 (link),38 (link)] and uses a dye selectively imported by cells that are undergoing apoptosis. Necrotic cells cannot retain the dye and, therefore, are not stained. HREC cells were treated as previously described in the “cell culture and treatment” section and the apoptosis assay was performed at day 6 of treatment in 96-well black plates (BD Falcon). The APOPercentage dye 3,4,5,6,-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein was added to each well (dilution 1:10) and cells were incubated for 30 more min at 37 °C in a cell incubator. After thoroughly washing, 100 µL of APOPercentage dye release reagent was added to each well, and the cell-bound dye recovered into solution was measured using a GENios plus microplate reader (Tecan) with excitation and emission of 530 and 580 nm, respectively. Results were calculated as the means ± SD of five measurements and expressed as a percentage of untreated control cells.