Biomedical Engineering Reference
In-Depth Information
glycoprotein E2 and the human tetraspanin protein CD81 is a key event for inhibitor
targeting. A flowcytometric assay using human hepatoma cells and fluorescently
labeled anti-CD81 antibody (JS81) has been used to profile the potencies of the
compounds inhibiting the E2-CD8 interaction [ref. 11]. However, new foci of interest
in flow cytometry are also generated by the introduction of new concepts in biology
and the clinical sciences, such as the role of programmed cell death in disease
processes and the hierarchical nature of tumor cell populations imposed by cells with
stem-like qualities. Exploration of such concepts in screening activities has been
aided byRNA interference (RNAi) technology in areas including oncogene addiction/
oncogenic shock, cancer stem cell biology, lineage dependency, and the epithelial--
mesenchymal transition [12].
The continued application of the principles of cytometry is apparent in the
development of cytometric bead array technologies, nowwell established, but arising
from the historical association between microspheres and flow cytometry [13]. Bead
arrays have provided a method of quantifying soluble analytes using earlier systems
such as the Luminex FlowMetrix platform [13]. The multiplexed assay format now
has applications in various areas of biology including protein expression profiling,
cancer markers, cardiac markers, cellular signaling, cytokines, chemokines, growth
factors, endocrinology, isotyping, matrix metalloproteinases, metabolic markers,
neurobiology, and transcription factors/nuclear receptors [14]. Critical to bead
technology is quality control, and staining protocols are available for using the
amine-reactive CBQCA dye to measure the amount of protein, without binding
partners, on the surface of a microsphere [15]. The high-speed spectral analysis of
individual particles in flow is set to enable new applications [16]. The measurement
of fluorescence and Raman spectra of individual particles at high speeds has been
performed on a modified Union Biometrica COPAS Plus instrument allowing red
excitation and optical fiber-based light collection and spectral analysis using a
spectrograph and a CCD array detector [16].
The application of the principles of cytometry is apparent in the development of
microscale devices and in vivo cytometry. An example of the latter relates to the
problem of cell assessment in lymphatics. Galanzha et al. have described how cells in
living animals can be counted by laser generation of photoacoustic signals in
individual cells, exploiting the principles of flow cytometry through a natural
“cell-focusing” phenomenon found in lymph vessels [17]. Thus, there is every
expectation that the involvement of flow cytometry in drug discovery will expand
and that it will be indispensable in new areas such as cell-based therapeutics from
discovery to quality control for regulatory compliance. Critically for drug discovery
applications there has been a drive to make advanced instrumentation user-friendly
and to remove the burden of expert knowledge required to extract reliable and robust
data. However, in parallel, flow cytometry has widened its attractiveness for the
development of novel reagents, requiring increased support from technology provi-
ders to navigate new applications to appropriate platforms.
In addressing cellular heterogeneity, flow cytometry and single-cell analyses in
general start to meet the aspiration to acquire systems level insights from the
molecular to the whole organism level in describing disease processes and revealing
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