DAPI Method: A Novel Assay to Evaluate the In vitro Impact of Nanomaterials on Mammalian Cells
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0951-E06-38
DAPI Method: A Novel Assay to Evaluate the In vitro Impact of Nanomaterials on Mammalian Cells Pavan M. V. Raja1, Jennifer Connolley2, Pulickel M. Ajayan3, Omkaram Nalamasu3,4, and Deanna M. Thompson2 1 Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180 2 Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180 3 Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180 4 Center for Integrated Electronics, Rensselaer Polytechnic Institute, Troy, NY, 12180
ABSTRACT The increasing importance of nanomaterial-related applications has given rise to concerns pertaining to their impact on human health. In vitro mammalian cell-based assays can provide a quick and simple estimate of the possible adverse effects of the nanomaterials. However, recent studies have questioned the efficacy of traditional assays such as the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, in evaluating cell-nanomaterial interactions, implying the need for alternate methods. We applied image analysis to enumerate the DAPI (2-[4-(Aminomethyl) phenyl]-1H-indole-6-carboximidamide, dihydrochloride) – stained cellular nuclei. Image analysis, being non-destructive, capable of automation, and applicable over a wide range of cell seeding densities, offers several advantages compared to older methods like the MTT assay and hemocytometry. Using image analysis, the impact of singlewalled carbon nanotubes (SWNT) on rat aortic smooth muscle cell (SMC) growth kinetics, were examined. Despite the carbon nanomaterial presence, the fluorescent signal from the nuclei was not noticeably impacted over the SWNT range examined (0.00-0.10 mg/ml). We anticipate that this method can also be applied to evaluate the biological impact of other nanomaterials. INTRODUCTION The rising popularity and potential of nanomaterial-based research, applications and industries gives rise to an urgent need to better understand their interactions with, and impact on human health and the environment. Carbon nanotubes (CNT), a sub-class of nanomaterials, with diverse application potential ranging from polymer nanocomposites to nanostructured therapeutic devices, can be considered as a model system to study the interactions between nanomaterials and biological systems, especially from a toxicology viewpoint. Several studies have addressed the effect of CNT (as-produced) [1-2], and functionalized with different chemical entities) [3-4] in vitro, on mammalian cells, and in vivo [5-6], in select animal models. The in vitro studies, which usually involve quantifying cell numbers or cell viability, constitute a rapid and economical means to evaluate the biological impact of CNT. Traditional in vitro approaches, such as hemocytometry [2-3], and the MTT assay [2] have been applied, to enumerate cells, in relation to their interactions with the CNT. However, these methods have certain disadvantages, which can be overcome by image analysis of fluorescently labeled nuclei. DAPI, (2-[4(A
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