title: Non-invasive and non-destructive measurements of confluence in cultured adherent cell lines.
abstract: 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.
authors.affiliation: Department of Histopathology, Trinity College Dublin, Central Pathology Laboratory, St. James's Hospital, Dublin 8, Ireland ; Department of Obstetrics and Gynaecology, Trinity College Dublin, Trinity Centre, St James's Hospital, Dublin 8, Ireland.
authors.forename: Steven
authors.initials: S
authors.lastname: Busschots
authors.affiliation: Department of Obstetrics and Gynaecology, Trinity College Dublin, Trinity Centre, St James's Hospital, Dublin 8, Ireland.
authors.forename: Sharon
authors.initials: S
authors.lastname: O'Toole
authors.affiliation: Department of Histopathology, Trinity College Dublin, Central Pathology Laboratory, St. James's Hospital, Dublin 8, Ireland ; Department of Obstetrics and Gynaecology, Trinity College Dublin, Trinity Centre, St James's Hospital, Dublin 8, Ireland.
authors.forename: John J
authors.initials: JJ
authors.lastname: O'Leary
authors.affiliation: Department of Histopathology, Trinity College Dublin, Central Pathology Laboratory, St. James's Hospital, Dublin 8, Ireland ; Department of Natural Sciences, Hendon Campus, Middlesex University, London NW4 4BT, United Kingdom.
authors.forename: Britta
authors.initials: B
authors.lastname: Stordal
keywords: Area Fraction Output Attached Cell Confluency; Area fraction; Cell count; Confluence; ImageJ; Non-destructive; Phase-contrast microscopy