Biomedical Engineering Reference
In-Depth Information
FACS methods are being applied to a wide range of stem cell research areas. For
example, a methodology has been evolved based on the availability of blue, cyan, and
yellowish-green fluorescent reporters (based on the jellyfish Aequorea victoria green
fluorescent protein) and the red fluorescent protein from a Discosoma coral. Flow
cytometric procedures based on such “rainbow” markers permit simultaneous
measurements of transgene expression and phenotypic discrimination of hemato-
poietic cell subsets is set to aid hematopoietic stem cell transduction protocols for
directed gene therapy [142]. Cytometric assays are being used to improve stem cell
tests to study embryotoxicity in vitro in a drive toward high-throughput screening of
compounds [143]. For example, Buesen et al. have recently reported an improvement
to the embryonic stem cell test (EST) aimed at revealing teratogenic potency in a
developing heart tissue. Quantitative marker protein flow cytometry (FACS-EST) for
sarcomeric myosin heavy chain and alpha-actinin expression could be performed
at an early point (7 days in mice) in development with a similar sensitivity for
embryotoxic potency as that achieved by the morphological end point of beating cell
agglomerates assayed at 10 days [144].
2.5.4 Cell Signaling
GPCRs have been proven to be the largest family of druggable targets in the human
genome. Receptor function operates through two primary processes, namely,
G-protein and beta-arrestin signaling. Recent insight into the operations of GPCRs
(seven-transmembrane (7TM) receptors) has led to the appreciation that certain
ligands initiate only portions of the signaling mechanism mediated by a given
receptor. This understanding is promoting a return to whole system assays and opens
new opportunities for drug discovery [145]. Flow cytometry has been used to select
clones with desired pharmacological profiles for use with the Tango 7TM receptor
assay technology, a high-throughput homogeneous assaymethod for monitoring beta-
arrestin recruitment that uses a live cell fluorescent readout [146]. Interestingly,
biosensor-centered label-free cell assay technologies are becoming a very active area
of effort for GPCR screening [147].
RGS proteins are important components of signal transduction pathways initiated
through GPCRs. The interaction of these regulators of G-protein signaling proteins
with the G-protein Galphao has been assessed using a flow cytometry protein
interaction assay (FCPIA). This approach permits the measurement of nanomolar
binding constants of such protein-protein interactions and Roman et al. have shown
the feasibility of targeting RGS/Galpha protein-protein interactions with small
molecules to modulate GPCR-mediated signaling processes [148]. The study used
a Luminex 96-well plate bead analyzer and a novel flow cytometric protein interaction
assay. To increase throughput, a set of 8000 compounds was compressed by
combining 4 compounds in a single assay well [149].
The signal transducers and activators of transcription (STATs) family responds to
a variety of cytokines, hormones, and growth factors. STATs are activated by
tyrosine phosphorylation, which results in their dimerization and translocation
into the nucleus where they exert their effect on transcription of regulated target
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