RECENT ADVANCES IN FLOW CYTOMETRY: PLATFORMS, TOOLS, AND CHALLENGES FOR DATA ANALYSIS - Flow Cytometry in Drug Discovery and Development - page 35

2.5 REAGENTS AND APPLICATIONS
A wide range of information sources exist for the selection of molecular probes,
matched light sources, and optical filter requirements [97]. Exploitation of the
functionality of particular fluorochromes, in terms of cellular penetration, compart-
mentalization, and affinity for cellular components, is a well-established approach in
flow cytometry. In high-content image-based screening assays, there is a demand for
segmentation with respect to major cellular compartments to allow translocation of
specific signals to be assessed through algorithm-driven routines. Here we consider
some selected areas in particular recent aspects of nanoparticle exploitation and cell
analyses relevant to drug discovery and therapeutics.
2.5.1 Micro- and Nanoparticles
Cell separations predominantly rely upon labeled cell migration within a magnetic
field termed magnetic-activated cell sorting (MACS) providing flow-through,
immunomagnetic cell separation. Antibodies reacting with specific cell surface
markers can be bound to magnetic beads and used to specifically capture cells
exhibiting the marker. Theoretical calculations showed that the throughput can be
increased to 10 6 cells/s by a scale-up of flow-through systems incorporating cell
sorting in a quadrupole field [98-100]. Differences inmagnetically induced motion of
diamagnetic, paramagnetic, and superparamagnetic microparticles are detected by
cell tracking velocimetry [101].
There are a number of different fluorescent probes and labels that are commonly
used for detection and tracking of a fluorescence signal from the cells, many of which
are organic fluorophores. The advantages of using nanoparticles in place of traditional
organic fluorophores have been widely reported [102, 103] with an impact on
diagnostics [104]. Advances in detector technology have also enabled single-
molecule imaging using quantum dots [105]. First, they are photostable allowing
long-term labeling of live cell populations [106]. Second, nanoparticles present major
advantages over conventional organic fluorophores as they are chemically stable and
are not metabolized by the cell. Quantum dots (eight colors) have been used for
multicolor immunophenotyping, where with conventional fluorophores at least 17
emission windows have been identified to reveal the phenotype of multiple antigen-
specific T-cell populations, hence setting the gold standard for polychromatic flow
cytometry [107].
An optical cell coding approach has been described by Machleidt et al. using
quantum dot encoded reporter cell lines to enable multiplexed reporter cell-based
assay with a beta-lactamase reporter gene readout as demonstrated for the analysis of
tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and interferon gamma
(IFN-gamma) [108]. Sukhanova et al. described the first application of nanocrystal-
encoded microbeads to clinical proteomics [109]. Two-color complexes, consisting
of encoded, antigen-covered beads, anti-antigen antibody or clinical serum samples,
and dye-tagged detecting antibodies, were analyzed by flow cytometry exhibiting
the narrow distributions of fluorescence signals, thus obviating the need of any