title: Flow cytometry evaluation of in vitro cellular necrosis and apoptosis induced by silver nanoparticles.
abstract: Particles possess unique properties in the nanoscale, e.g., enhanced catalytic activity, high surface area, and light emission/absorption properties, that might result in interference with colorimetric in vitro cytotoxicity assays such as MTT, XTT or MTS. Alternatively, assays that do not use spectrophotometric detection, such as trypan blue exclusion or flow cytometry (FC) based assays, are less likely to be influenced by nanoparticle interference. The aim of this study was to evaluate FC assays to assess the cytotoxicity of three different sizes (10, 100, or 200 nm) of silver nanoparticles (AgNPs) at different mass concentrations (1, 25, or 50 ug/ml) in L-929 fibroblast cells. After 4 h and 24 h exposure, cell necrosis and apoptosis were assessed using 7-AAD and Annexin V dyes, respectively, with FC. The data indicate that cell necrosis and apoptosis in AgNP-exposed fibroblasts depends on dose, exposure time, and AgNP size. The data indicate that AgNPs produced a dose- and time-dependent decrease in cell viability; however, 10 nm AgNPs were significantly more toxic than larger-sized particles. Thus, standard FC assays can be utilized to assess apoptosis and necrosis in response to nanomaterial exposure.
authors.affiliation: Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD 20993, USA.
authors.forename: Girish
authors.initials: G
authors.lastname: Kumar
authors.affiliation: Office of Cellular, Tissue, and Gene Therapies, Center for Biologic Evaluation and Research, US Food and Drug Administration, Silver Spring, MD 20993, USA.
authors.forename: Heba
authors.initials: H
authors.lastname: Degheidy
authors.affiliation: Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD 20993, USA.
authors.forename: Brendan J
authors.initials: BJ
authors.lastname: Casey
authors.affiliation: Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD 20993, USA. Electronic address: Peter.Goering@fda.hhs.gov.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Goering
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D055598:Chemical Phenomena; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000737:chemistry; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D053768:Metal Nanoparticles / Q000633:toxicity*; D051379:Mice; D046529:Microscopy, Electron, Transmission; D009336:Necrosis / Q000188:drug therapy*; D009391:Nephelometry and Turbidimetry; D010316:Particle Size; D012212:Rheology; D012834:Silver / Q000633:toxicity*; D013499:Surface Properties; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D017304:Annexin A5; D005456:Fluorescent Dyes; D003609:Dactinomycin; D012834:Silver; C025942:7-aminoactinomycin D
keywords: Apoptosis; Cytotoxicity; Flow cytometry; Nanotoxicology; Silver nanoparticles