title: The Analysis of Cell Cycle, Proliferation, and Asymmetric Cell Division by Imaging Flow Cytometry.
abstract: Measuring cellular DNA content by conventional flow cytometry (CFC) and fluorescent DNA-binding dyes is a highly robust method for analysing cell cycle distributions within heterogeneous populations. However, any conclusions drawn from single-parameter DNA analysis alone can often be confounded by the asynchronous nature of cell proliferation. We have shown that by combining fluorescent DNA stains with proliferation tracking dyes and antigenic staining for mitotic cells one can elucidate the division history and cell cycle position of any cell within an asynchronously dividing population. Furthermore if one applies this panel to an imaging flow cytometry (IFC) system then the spatial information allows resolution of the four main mitotic phases and the ability to study molecular distributions within these populations. We have employed such an approach to study the prevalence of asymmetric cell division (ACD) within activated immune cells by measuring the distribution of key fate determining molecules across the plane of cytokinesis in a high-throughput, objective, and internally controlled manner. Moreover the ability to perform high-resolution, temporal dissection of the cell division process lends itself perfectly to investigating the influence chemotherapeutic agents exert on the proliferative capacity of transformed cell lines. Here we describe the method in detail and its application to both ACD and general cell cycle analysis.
authors.affiliation: Flow Cytometry Core Facility, Newcastle Biomedicine, Newcastle University, Newcastle-upon-Tyne, NE1 7RU, UK. andrew.filby@newcastle.ac.uk.
authors.forename: Andrew
authors.initials: A
authors.lastname: Filby
authors.affiliation: FACS Laboratory, London Research Institute, Sir Francis Crick Institute, 44 Lincoln's Inn Fields, Holborn, UK.
authors.forename: William
authors.initials: W
authors.lastname: Day
authors.affiliation: FACS Laboratory, London Research Institute, Sir Francis Crick Institute, 44 Lincoln's Inn Fields, Holborn, UK.
authors.forename: Sukhveer
authors.initials: S
authors.lastname: Purewal
authors.affiliation: Lymphocyte Interaction Laboratory, London Research Institute, Sir Francis Crick Institute, 44 Lincoln's Inn Fields, Holborn, London, WC2A 3LY, UK.
authors.forename: Nuria
authors.initials: N
authors.lastname: Martinez-Martin
mesh_terms: D000818:Animals; D060049:Asymmetric Cell Division; D002453:Cell Cycle; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D008214:Lymphocytes / Q000166:cytology*
keywords: Asymmetric cell division; Cell cycle; Cell proliferation; CellTrace Violet; Dye dilution; Mitosis