Biomedical Engineering Reference
In-Depth Information
fluorescence compensation [109]. A combination of fluorescence and magnetic
properties for nanobeads provides new applications in simultaneous bioseparation
and biosensing. Recently, Di Corato et al. have described spherical nanobeads
(30-400 nm) with a core magnetic domain of iron oxide sheathed in fluorescent
oligothiophene molecules engrafted to an amphiphilic polymer with properties
suitable for cell sorting applications [110].
Overall, there is continuing optimism over the applications enabled by the use of
engineered nanocrystals with studies suggesting that it may be possible to exploit a
cell
s active transport machinery for delivery to specific nuclear and cytoplasmic
compartments [111]. Inhibitor studies with flow cytometry have recently revealed that
the mechanism of fluorescent “nanodiamond” (FND) uptake is energy-dependent
clathrin-mediated endocytosis with further evidence for biocompatibility for cell
tracking [112].
2.5.2 Cell Death and Cell Cycle Arrest
Simple applications of flow cytometry aid clarification of cell responses to small
molecule screens. For example, the promotion of apoptosis in cancer cells remains an
attractive approach for targeted therapies [113] with multiple flow cytometric assays
being available. In a novel cell- and caspase-based Anticancer Screening Apoptosis
Program (ASAP) HTS assay, 4-aryl-4H-chromenes have been identified as potent
apoptosis inducers [114]. High-throughput flow cytometry has been applied in a
screen for apoptosis-activating proteins in transiently transfected cells. In this case,
the assay is based on the detection of activated caspase-3 with a specific antibody in
cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent
protein [115]. Cell cycle analyses where capture of cells into suspension is proble-
matic, high-content analysis (HCA), can be achieved by automatedmicroscopy-based
technologies such as the Cellomics
s ArrayScan reader, although in general multiple
descriptors of the cell cycle are required to aid reliable and accurate quantification
of cell cycle phases [116]. New nuclear/cytoplasmic discriminating probes have been
developed (DRAQ5 and CyTRAK Orange [117]) with spectral separation from the
commonly used reporter proteins (e.g., eGFP, YFP, mRFP) and fluorescent tags such
as AlexaFluor 488, fluorescein, and Cy2 [118]. DRAQ5 has recently found applica-
tion in tracking the pharmacodynamic responses to cell cycle perturbing agents [119,
120]. Clearly, there is amove toward assays in which there is retention of functionality
of cells and cross-platform analysis solutions. Here we outline a new approach (P.J.
Smith et al., unpublished data) for tracking cells undergoing long-term cell cycle
arrest using CyTRAK Orange in studies on chemoresistance and drug targeting
(Figure 2.2).
New solutions are being sought to the problem of inefficient drug delivery, a
contributor to tumor chemoresistance and drug failure. For example, enhancing tumor
access by depleting tumor-associated stromal tissue through the inhibition of the
hedgehog cellular signaling pathway has recently been proposed [121]. Indeed, it is
recognized that anticancer drugs are unable to penetrate into poorly oxygenated tumor
tissue masses. Furthermore, low oxygen concentration may in itself represent a key
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