Gain Critical Insights from Advanced Cell Models with Real-Time Analysis - 21

Real-Time Analysis of Advanced Cell Models | WHITE PAPER

Standard Techniques for Analyzing Cellular Subsets:
Strengths and Weaknesses
Flow cytometry is the best characterized and established
method for true cell-by-cell analysis. Cells are analyzed one by
one in a flow stream for light scattering and fluorescence. When
combined with labeled antibodies to cell surface markers and
fluorophores, a wide range of multivariate analyses can be applied. Each cell can be classified (binned) into a discrete subset
to quantify how many of each cell type are present, and what the
associated properties (e.g. cell health, size, etc.) of each subset
are. Using multiple fluorophore labels and high-end instrumentation, it's possible to create 10s of distinct subsets simultaneously
in a cell population (e.g. peripheral blood mononuclear cells,
or PBMCs). High-content imaging (HCI) methods can also be
applied to resolve individual cells for heterogeneity and subset
analyses. Typically, nuclear labels (e.g. DAPI) and advanced image
analysis algorithms are combined to identify single cells and their
boundaries and to extract features on a cell-by-cell basis. Again,
subsets can be analyzed separately based on gating criteria.
Mostly, this work is done using fix-and-stain protocols to maximize fluorescent signals to background and to permit labeling of
intracellular structures (e.g. organelles). The third main approach
is time-lapse microscopy. Here, researchers observe living cells
under closely controlled environmental conditions, typically with
single cell tracking and high spatial and temporal resolution.
Unlike the other approaches, this gives critical insight into cell
morphology, dynamics and cell/cell interactions. Together, these
three approaches provide an impressive toolkit for analysis at the
cell-by-cell level.
Nevertheless, there remains a significant unmet need for additional technical solutions in this area. For both flow cytometry
and HCI, the cell preparation and labeling methods cause significant disruption to the cellular environment potentially introducing experimental artifacts. HCI is suitable for analyzing adherent
cells but not non-adherent cells, while for flow cytometry the
converse is true. In both cases, the analytical approaches are only
21

| January, 2019

Cell segmentation

Fluorescence intensity

Verify with back-gated images

Features: phase and
fluorescence
Subset 1

View populations over time and classify cells

Figure 1: IncuCyte Cell-by-Cell analysis: concept and workflow

Plot subset time-courses

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Gain Critical Insights from Advanced Cell Models with Real-Time Analysis

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