Use of fluorescent Fab/Ab complexes and IncuCyte live-cell analysis to dynamically track cell surface markers and cell populations in mixed cultures

Abstract

Abstract Fluorescently-labeled antibodies are widely used for visualising cellular protein expression/distribution (e.g. immunocytochemistry) and immuno-phenotyping (e.g. flow cytometry). However, their applications are largely confined to end-point or short term (min-h) detection, and the cell processing and labeling steps that are required often perturb the biology of interest. To enable longer-term, fully kinetic applications of cell surface protein markers in living cells, we have developed a novel strategy based on fluorescently-labeled antibody fragments (Fabs) and IncuCyte live-cell analysis. An Fc-targeted anti-mouse Fab fragment conjugated to a green-emitting fluoroprobe (IncuCyte FabFluor-488) was used to tag Abs to surface markers (e.g. CD4, CD20, Her2) via a simple one-step, no-wash protocol. Addition of the corresponding FabFluor-488-Ab complex to living cells (e.g. Jurkats, RAMOS, SKOV-3) produced long lasting (up to 5d), specific and stable cellular fluorescence at relatively low Ab concentrations (1mg ml−1) that did not perturb cell morphology or growth (IncuCyte S3). To illustrate the application of this approach the method was used to (a) quantify upregulation of the checkpoint protein PDL-1 over 48h in IFN-gamma-treated MDA-MB231 and SKOV-3 and tumor cells, (b) identify cellular subsets in PBMCs, and (c) observe proximity and engagement of A549 target cells by CD-8 positive PBMCs in immune-cell killing experimentsThis FabFluor-488/IncuCyte method enables long-term tracking and quantification of cell surface protein expression and the ability to identify cell subsets in living cultures over time. This method should prove powerful in analyses on complex and advanced heterogeneous cell models.