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culture plates and grown to 100% confluence. Wounds were created by scraping the monolayer of cells with a sterile pipette tip, washed with PBS to remove the floating cells and incubated with fresh medium in the presence or absence of AgNPs-EPS. The images of scratched area were captured (at 40× magnification) using an inverted microscope equipped with digital camera immediately after wounding and after 6 and 24 h. The scratched area was calculated by Image J software. Gelatin zymography SKBR3 cells (5 × 104 cells/well) were seeded on 6-well plates, grown to 90% of confluence and treated for 24h with AgNPs-EPS IC50 in a serum-free medium. Conditioned media were collected, centrifuged to remove cellular debris, dialyzed and lyophilized. Equal amounts of total proteins (5 μg) were separated by electrophoresis, on a 7.5% sodium dodecyl sulfate (SDS)–polyacrylamide gel containing 0.1% gelatin, under non-reducing conditions. After electrophoresis, the gels were washed for 1h with a buffer containing 50 mM Tris-HCl, pH 7.5 and 2.5% Triton X-100, to remove the SDS and then incubated with activation buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, for 18h at 37 °C. Gel was stained with Coomassie Brilliant Blue G 250 and de-stained with H2O milliQ. The bands intensity were measured with Image J software. Morphological assessment and Acridine orange/ Ethidium bromide (AO/EB) SKBR3 cells were seeded in 12 well culture plates with a cell density of 1 × 105 cells/well. After 24 h cells were incubated for 24 h with the IC50 concentration of the AgNPs-EPSaer, and observed for morphological changes assessment under a phase contrast inverted microscope (Carl Zeiss) at 400X magnification. For AO/EB, the cells were seeded at the same conditions in a cover slip and after treatments, cells were washed twice with PBS and stained for few minutes with 200 μL of the Acridine Orange (100μg/mL), Ethidium Bromide (100μg/mL) mixture (1:1, v/v). Cells were immediately observed under a fluorescent microscopy (Carl Zeiss) at 630X magnification. Evaluation of ROS generation Intracellular ROS levels were measured by using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH- DA) and Dihydroethidium (DHE) assays. DCFH-DA is routinely used to measure intracellular generation of H2O2 and other oxidants; it is a cell-permeable probe, hydrolyzed intracellularly to the DCFH. Two- electron oxidation of DCFH results in the formation of a fluorescent product, dichlorofluorescein (DCF). DHE is another widely used probe for detecting intracellular O2-.The red fluorescence formed from the two-electron oxidation product, is usually equated to intracellular superoxide formation. SKBR3 cells were plated in 96 well plates at a density of 5 × 103/well, allowed to growth overnight and incubated for 24 h with different AgNPs- EPSs concentrations. At the end the medium was replaced with the culture medium containing DCFH-DA (10 μM) and DHE (10 μM) and incubated for 30 min at 37°C. Then the medium was replaced with PBS and the fluorescence intensity was analyzed by spectrofluorimeter with an excitation of 488 nm and emission wavelength of 525 nm for DCFH-DA and an excitation of 540 nm and emission wavelength of 590 nm for DHE. Data normalization was performed with parallel MTT assay. DNA extraction and flow cytomety DNA was extracted from SKBR3 treated cells as previously described [73], with some modifications. The cells were detached from the flasks with trypsin and suspended in 0.5 ml of TE buffer (100 mM Tris-HCl, 100 mM EDTA pH 8). 50 μl of 10% SDS and 25 μl of 1 mg/ml Proteinase K were added to the cell suspension and incubated for 30 minutes at 55°C. Nucleic acids were extracted using phenol-chloroform, precipitated with 1/10V 3M Na-acetate and 2V absolute ethanol and resuspended in 100 μl TE buffer. The same quantity of extracted DNA was loaded on a 1% agarose gel prepared in 0.5X TE buffer containing gel red. Gel was visualized under UV light using a Bio-Rad Trans illuminator and photographed by using a Polaroid camera. Apoptosis of SKBR3 cells after treatment with IC50 of AgNPs-EPS for 24 h was analyzed by flow cytometry essentially as described by Riccardi [74]. Briefly cells were fixed in 70% ethanol, washed in PBS and resuspended in DNA extraction buffer (200 mM Na2HPO4, 0.1% Triton X-100). After staining with Propidium Iodide (1μg/mL) for 30 min, fluorescence intensity was acquired in the FL2 channel by flow cytometry on a FACSCalibur flow cytometer (BD, New Jersey, USA). Data acquisition was performed with CellQuest Pro (BD) software, and analyzed with WinList software (Verity Software House, Topsham, USA). Fluorescence staining with Acridine Orange (AO), monodansylcadaverine (MDC) and Lysotracker® SKBR3 cells were grown on coverslips and treated for 24 h with the IC50 concentration of AgNPs- EPSaer. Acidic vesicular organelles (AVOs) were labeled with 1 μg/ml of Acridine Orange, autophagosomes and autolysosomes with 0.05 mM of MDC (Sigma, St. Louis, MO, USA) while acidic compartments were labeled by incubating the cells with 75 nM of LysoTracker® (Molecular Probes, Life Technologies) in the culture www.impactjournals.com/oncotarget 9699 OncotargetPDF Image | Anticancer activity of biogenerated silver nanoparticles
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