Data Availability StatementAll relevant data are within the paper. can be used as a solid inhibitor for a wide Nobiletin cell signaling spectral range of antimicrobial actions, such as for example those of bacterias, fungi, and Nobiletin cell signaling infections. Compared with additional metals, metallic displays higher toxicity to microorganisms while exhibiting lower toxicity to mammalian cells[1]. It’s been conformed that Ag+ ions, a prototypical antimicrobial metallic species by means of a metallic nitrate option, are energetic against an array of bacterias and fungi[2]. Nanometer-sized metallic particles (AgNPs) possess long been recognized Nobiletin cell signaling to come with an antibacterial impact. AgNPs are smaller sized than 100 nm generally, including 20C15,000 metallic atoms, and show unusual physical, chemical substance and natural properties[3]. Because of the strong antibacterial actions, the usage of AgNPs and their composites continues to be suggested for avoiding infection in medical procedures[4], in the coatings of medical products[5,6] or like a water disinfectant or space aerosol[3] even. However, the systems of the antibacterial impact are unclear. The most common system of AgNPs may be the inhibition of the enzymatic function of some proteins by interaction with the thiol RPD3L1 groups of L-cysteine [7C9]. Promoting the permeability of the bacterial membrane [1] and disrupting the membrane integrity [10] are also thought to be responsible for the antibacterial effect. Moreover, it has been discovered that silver can bind to the DNA, increasing the decomposability of genome DNA[11C13] or inactivating the respiratory chain, inducing the formation of hydroxyl radicals[9]. In previous studies, the antibacterial mechanism of AgNPs has only been partially elucidated. Programmed cell death (PCD), which induces apoptosis, is an essential mechanism in eukaryotic organisms[14] and also can been found in prokaryotes cells, such as cells [15]. In our work, a new mechanism of the antibacterial activity of AgNPs was identified. For the first time, we demonstrate the antibacterial mechanism of AgNPs in terms of inducing bacterial apoptosis. Materials and Methods Reagents and antibodies AgNP solution 100AGS-WMB1000C (diameter: 5~10 nm, concentration: 1000 ppm) was purchased from Shanghai Huzheng Nanotechnology Co., Ltd. The propidium iodide (PI) reagent (50 g/ml) was purchased from BD Co. Bovine serum albumin (BSA) was produced from Sigma Co. The FITC-conjugated annexin V and PI Nobiletin cell signaling kit was obtained from Dojindo Molecular Technologies, Inc. The cell proliferation kit was purchased from Roche Co. All other chemicals were supplied by Aldrich and used as received. The strain (ATCC 25922) was purchased from American Type Culture Collection (ATCC) and conserved in our laboratory. The FACS buffer was prepared with 0.5% BSA, 2 mM EDTA and 500 ml PBS. Luria-Bertani (LB) liquid medium and solid medium were prepared in our laboratory. Nanoparticle characterization by TEM The morphology of the AgNPs was characterized by an analytical transmission electron microscope (TEM). Aliquots of the AgNP solutions (5 and 10 g/ml) were dropped onto the carbon-coated copper (Cu) grid and then air-dried before TEM observation. The chemical analysis of the AgNP solutions was performed using the energy dispersive x-ray spectroscopy (EDX) module attached to the TEM (JEOL JEM-2100). Antibacterial effect of AgNPs measured Nobiletin cell signaling by OD600 and CFU The cells were cultured in 5 ml of LB medium at 37C overnight. After incubation, the cells were diluted (1:100) in 300 ml of LB medium and incubated with 5 or 10 g/ml AgNPs at 37C and 220 rpm for 24 h. The bacterial concentrations were determined by both measuring the optical density (OD) and counting colony-forming units (CFU). The absorbance was determined at 600 nm by spectrophotometry (Beijing Purkinje General Instrument Co., Ltd., China). Each experiment was performed twice, and the growth curves were plotted by Prism 5 software (http://www.graphpad.com/). Flow cytometry analysis of dead bacteria The cells were cultured overnight and incubated with 300 ml of LB (under 1:100 dilution) including 5 or 10 g/ml AgNPs for 1, 2 and 3 h. At every time stage, the cells had been spun down at 10000g for 10 min and resuspended in.