Biomedical Engineering Reference
In-Depth Information
15.4.3 Combining FCB and High-Throughput Autosamplers
Moving forward, we anticipate that FCB will be used together with high-throughput
autosamplers, such as the plug flow platform, to enable ultrahigh throughput screen-
ing by flow cytometry. Combining a relatively “simple” FCB matrix that encodes 9
samples (a 3
3 matrix) with a high-throughput autosampler system would allow
nearly 100,000 compounds to be screened in a day. At this point, the bottleneck would
likely rest not on sample acquisition on the cytometer, but rather with drug treatment,
sample preparation, and staining. Therefore, flow cytometers can clearly manage the
sample throughput necessary to screen large compound libraries. What is perhaps
more important is development of cell-based assays that are robust at large scales and
minimization of antibody costs. The combination of assay optimization and hardware
configurations makes it feasible to perform advanced cell-based assays on a com-
pletely new scale.
15.5 SECONDARY SCREENING AND PRECLINICAL TESTING
Once lead compounds have been identified via high-throughput screens such as in vitro
kinase assays, translocation, or gene reporter assays, flow cytometry provides an
excellent platformformore fully elucidating compound selectivity and toxicity, aswell
as monitoring compound effects in vivo in preclinical animal models. In particular,
phospho flow is perfectly suited to drug discovery targeting signaling proteins such as
kinases, phosphatases, and transcription factors [2, 16]. The multiparameter capabil-
ities of phospho flow enable the researcher to evaluate multiple components of the
same signaling pathway or key members of multiple different signaling pathways to
characterize selectivity in cell-based systems. Then, once a suitably selective com-
pound has been found, phospho flow provides a platform for analyzing compound
efficacy directly in peripheral blood from preclinical models of disease. This ability to
use the same assay platform throughmultiple steps of the discovery process reduces the
time needed to validate compounds and eliminates the requirement for completely
distinct metrics to monitor drug efficacy in vivo. In this section, we will discuss the use
of phospho flow in kinase drug screening and how valuable insight is gained by
analyzing drugs in the context of a signaling network, not individual proteins.
15.5.1 Cell-Based Screening
Our laboratory has a strong interest in understanding how the immune system
functions in the context of inflammation, bacterial challenge, antigen challenge, and
autoimmune diseases such as rheumatoid arthritis and SLE [7, 8, 17-19]. In multiple
disease states, such as cancer and SLE, we have found that modulation of cytokine
signaling plays a central role in the disease and correlates with disease severity and
patient outcome.
Cytokines, small proteins that bind to specific cell-surface receptors, play a
central role in nearly all immunological system challenges as the major means of
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