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Steven Busschots; Sharon O’Toole; John J. O’Leary; Britta Stordal (2015)
Publisher: Elsevier
Journal: MethodsX
Languages: English
Types: Article
Subjects: Area Fraction Output Attached Cell Confluency, Confluence, ImageJ, Science (General), Phase-contrast microscopy, Q1-390, Biochemistry, Genetics and Molecular Biology, Non-destructive, Cell count, Confluence; Cell count; Phase-contrast microscopy; ImageJ; Area fraction; Non-destructive, Area fraction
PUBLISHED Many protocols used for measuring the growth of adherent monolayer cells in vitro are invasive, destructive and do not allow for the continued, undisturbed growth of cells within flasks. Protocols often use indirect methods for measuring proliferation. Microscopy techniques can analyse cell proliferation in a non-invasive or non-destructive manner but often use expensive equipment and software algorithms. In this method images of cells within flasks are captured by photographing under a standard inverted phase contract light microscope using a digital camera with a camera lens adaptor. Images are analysed for confluence using ImageJ freeware resulting in a measure of confluence known as an Area Fraction (AF) output. An example of the AF method in use on OVCAR8 and UPN251 cell lines is included. • Measurements of confluence from growing adherent cell lines in cell culture flasks is obtained in a non-invasive, non-destructive, label-free manner. • The technique is quick, affordable and eliminates sample manipulation. • The technique provides an objective, consistent measure of when cells reach confluence and is highly correlated to manual counting with a haemocytometer. The average correlation co-efficient from a Spearman correlation (n = 3) was 0.99 ± 0.008 for OVCAR8 (p = 0.01) and 0.99 ± 0.01 for UPN251 (p = 0.01) cell lines. This study was funded by a PhD Scholarship from the Royal City of Dublin Hospital Trust (S.B.) and the Emer Casey Foundation (S.B) as part of the DISCOVARY Consortium, an Irish Cancer Society Postdoctoral Fellowship (B.S.) and a Marie Curie Re-integration Grant from the European Union P7 programme (B.S.).