Abstract

Here we describe a novel labelling and analysis
strategy to enable long-term, non-invasive quantification of immune cells based
on IncuCyte® live-cell imaging.

An Fc-targeted anti-mouse Fab fragment conjugated
to a green-emitting fluoroprobe (IncuCyte FabFluor-488) was used to tag
antibodies to cell surface markers. Addition of the FabFluor488-antibody
complex to living cells, including OptiGreen background suppressor, produced fluorescent labelling that was bright and stable
without perturbing cells. With new image analysis and visualisation tools,
individual cells were segmented from the phase image and quantified cell by
cell for fluorescence. In PBMCs, anticipated frequencies of lymphocyte
subpopulations (CD4, CD8) were detected using this method. Cell subsets can be
classified for analysis determining dynamic changes in response to stimuli.

This method is powerful in analyses on dynamic
heterogeneous cell models and cell-cell interactions.