Page 74 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Live-cell Immunocytochemistry

Long-term tracking and quantification of cell surface protein markers

Introduction

Immunocytochemistry (ICC) is a powerful laboratory technique for able to monitor their activity in real-time and in their natural
visualizing the cellular and subcellular location of proteins using environment is paramount. Consequently, there is a strong unmet
fluorescent-labeled antibodies (‘immunofluorescence’). The study need for technical solutions that can allow the tracking of protein
of cell protein expression, and the modulation of it, is a crucial distribution and abundance over time in living cells, and link these
method used to understand gene expression, and ultimately the vto cell morphology and function.
biology of the cell. With cell fixation protocols, labeled antibodies
and specialized microscopes, spatial resolution down to the In this chapter, we illustrate how kinetic, live-cell analysis of
nanometer level can be achieved to evaluate the expression surface protein expression using fluorescently-labeled antibodies
of proteins in the nucleus, specialized organelles and the cell opens up new possibilities for connecting long-term dynamic
membrane. changes in protein abundance and distribution in cells with
morphology and function.
Where conventional ICC is less useful, however, is in studying
dynamic changes in protein expression over time and under IncuCyte live-cell immunocytochemistry
®
physiologically relevant conditions. The technique includes a
number of lengthy and time-consuming steps – fixing, washes, at a glance
and staining – which can take anything between six and 24 hours.
These procedures can also lead to physical loss of cells, or result In order to measure the dynamic changes in cell surface protein
in unhealthy cells that are losing their viability because of fixation expression while simultaneously monitoring morphological
and are unlikely to give an accurate picture of the biology of changes, IncuCyte® FabFluor-488 Antibody labeling reagents
interest. are combined with Fc-containing antibodies. These conjugated
FabFluor-Ab complexes are then added directly to live-cells in
ICC is a widely used technology for studying changes in protein a single-step mix and read assay. Time-lapse images of cellular
surface molecules – the key communicators that dictate how a fluorescence can then be gathered over hours and days, and
cell responds to external stimuli and changes its shape, protein automatically analyzed to provide an index of the levels and
expression and function in response. These cell surface molecules pattern of expression over time. An overview of the assay
are the targets of many existing and future drugs and being workflow is shown below.

1 Seed cells 2 Label test antibody 3 Add incucyte 4 Add labeled AB 5 Live-cell
opti-green
fluorescent imaging

Cell seeding Labeling of test antibody IncuCyte® Opti- IncuCyte® FabFluor-488- Automated imaging and
Seed cells (50 μl/well, with IncuCyte® FabFluor-488 Green background labeled antibody addition quantitative analysis
5-30K/well) into 96-well reagent suppressor addition Add antibody-FabFluor Capture images, (time
plate. Mix antibody and FabFluor-488 Add 50 μl/well, (3x mix (50 μl/well) to cell span and objective
NOTE: for non-adherent reagent at a molar ratio final concentration). plate. Non-adherent cells depends on assay and
cell types, PLO coat plate of 1:3 in media, 3x final – spin plate cells type, 10x or 20x) in
prior to cell seeding. concentration. Incubate for 15 IncuCyte.
minutes to allow conjugation.
Figure 1. Overview of IncuCyte® live-cell immunocytochemistry workflow.

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