Silver ag nanoparticles

For the alkaline single-cell gel electrophoresis (Comet assay) procedure [15] , 3 × 10 5 NHK or NHDF were incubated with nanoparticles 0.05% [w/v] for 24 h, harvested and re-suspended with 1% low-melting point agarose. 50 cells per slide and 3 slides per treatment were examined by Lucia Comet Assay Software TM (Lucia Cytogenetics, Prague, Czechia). DNA damage was expressed as the relative tail length.
Intracellular reactive oxygen species (ROS) levels were determined by 6-Carboxy-2´,7´dichlorodihydrofluorescein diacetate (H2DCFDA) assay [24] . NHK and NHDF at a density of 5 × 10 4 cells were incubated for 1 h with nanoparticles 0.05% [w/v] in KGM or DMEM without FCS, respectively, and after washing incubated with 25 µM H2DCFDA for 1 h and analyzed on a FACSCanto II (BD Biosciences, Heidelberg, Germany). FlowJo software (Treestar, Ashland, OR, USA) was used for data analysis. Dead cells and debris were excluded by forward/sideward scatter plot.
For the comet assay and ROS determination, PBS (10%) in KGM or DMEM without FCS, respectively, served as negative control, silver (Ag) nanoparticles (diameter 40 nm, 5 µg/ml, Sigma-Aldrich) served as positive control.