Biomedical Engineering Reference
In-Depth Information
account the increase in cellular biomass that accompanies unresolved cell cycle
interruption and here we describe the application of a novel dye, CyTRAK Orange
(CYO, Biostatus Ltd, UK), capable of staining live cells (e.g., leukocytes and
epithelial cells). It was developed as a cell-masking agent for the differentiation of
cell features in HTS imaging applications. In flow cytometry, CYO has the advantage
of not being excited by a 638 nm laser, allowing its use with APC, APC conjugates,
and PE-Cy7 and analogues, but importantly provides a convenient integral of cell size
and DNA content. Figure 2.2 shows the impact of hypoxia-mediated AQ4N conver-
sion to a cell cycle arresting agent in human A549 lung cancer cells tracked using side
scatter and CYO fluorescence. Convenient methods of identifying such events in live
cell preparations permit sorting or additional functional assays to be performed with
resolution for arrested and nonarrested subpopulations.
2.5.3 Multidrug Resistance and Cancer Stem Cells
It is now well recognized that the ATP binding cassette (ABC)-containing family of
proteins has a pharmacological impact on efficacy of many drugs in current use [129].
High-throughput flow cytometry provides a sensitive functional approach for the
assessment of selective inhibitors of key transporters such as ABCB1, ABCC1, and
ABCG2 [130]. Different computational methods and models have been evolved to
predict ABC transporter substrate properties of drug-like compounds with the
potential for pharmacological profiling of compound series, in particular their
ADME/Tox properties [131]. The P-glycoprotein, encoded by the ATP Binding
Cassette B1 (ABCB1) gene, contributes tomultidrug resistance (MDR) and continues
to be subject to a search for novel inhibitors. Using ABCB1 overexpressed T-ALL cell
line, a flow cytometry-based high-throughput screening (HTS) assay has been used
to assay ABCB1-mediated doxorubicin efflux, revealing mometasone furoate as a
previously unrecognized ABCB1 antagonist [132].
Cancer stem cells (CSCs) may contribute to chemoresistant subfractions in a
variety of malignancies [133-135] and underpin the post-therapeutic recovery
potential of tumors. Enhanced drug efflux, via ABC transporters, has been linked
with CSC biology [135, 136]. The ABCG2 transporter (MXR/BCRP/ABCP1) shows
an intriguing link with the CSC phenotype [135] in contributing to the flow cytometric
definition of the pluripotential “side population” (SP) [137, 138]. SP cells are capable
of efficient exclusion of the DNAminor groove-binding cytotoxic dye Hoechst 33342
as detected by changes in the UV-excited fluorescence emission spectrum of nuclear
bound dye [139]. Hoechst 33342 SP analysis is a commonmethod for identifying stem
cells in mammalian hematopoietic and nonhematopoietic tissues. Although widely
employed for stem cell analysis, a significant drawback of SP analysis has been the
expense of UV laser sources, although benchtop analyzer (the Quanta Analyzer, NPE
Systems) and platforms using nonlaser UV sources have been described [140]. An
alternative is the identification of Hoechst 33342 surrogate molecules with similar
pump specificity as Hoechst 33342, but with better violet excitation characteristics
(e.g., the DyeCycle Violet reagent (excitation peak 369 nm)), permitting side
population analysis on flow cytometers with violet lasers [141].
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