title: Best practices for application of attachment cells to in vitro micronucleus assessment by flow cytometry.
abstract: This work seeks to provide users with guidance on cell culture, treatment, processing and analytical conditions for achieving optimal performance of the in vitro micronucleus assay using the In Vitro MicroFlow(®) method. Experimental data are provided to support the advice described. The information provided covers specific topics or issues that are identified as critical to the methodology and thus is meant to work with instruction manuals, published papers and other references, and not as a replacement for these documents. The content is divided into several sections. Cell culture and treatment describes conditions for routine maintenance of cells as well as treatment with test articles. Preparation and processing of samples details steps found to be critical in execution of the procedure. Instrument parameters and analysis covers set-up of the flow cytometer and evaluation of the samples. General assay considerations and interpretation of results describes examination of data in terms of assay validity, viability and genotoxicity assessment. The goal is to educate users and enable them to design, conduct and interpret flow cytometric in vitro micronucleus (MN) studies. Readers should obtain an understanding of specific cell culture practices, options for assay formatting and execution and the information required to successfully integrate and validate the in vitro MN assay into their existing safety program.
authors.affiliation: Litron Laboratories, Rochester, NY, USA. Electronic address: jbemis@litronlabs.com.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Bemis
authors.affiliation: Litron Laboratories, Rochester, NY, USA.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Bryce
authors.affiliation: Bayer HealthCare, Investigational Toxicology, Wuppertal, Germany.
authors.forename: Marlies
authors.initials: M
authors.lastname: Nern
authors.affiliation: Bayer HealthCare, Investigational Toxicology, Berlin, Germany.
authors.forename: Marian
authors.initials: M
authors.lastname: Raschke
authors.affiliation: Bayer HealthCare, Investigational Toxicology, Berlin, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Sutter
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001386:Azides / Q000633:toxicity; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D006224:Cricetinae; D004249:DNA Damage / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D015162:Micronucleus Tests / Q000379:methods*; D009336:Necrosis / Q000139:chemically induced / Q000473:pathology; D013936:Thymidine / Q000633:toxicity
chemical_list: D001386:Azides; C014535:8-azidoethidium; D013936:Thymidine
keywords: Attachment cells; Best practices; Flow cytometry; In vitro micronucleus