Biomedical Engineering Reference
In-Depth Information
flowcytometer relative to the placement ofHyperCyt.As notedabove, acquisition time
for individual plates must be coordinated with the throughput of plate preparation.
4.5.4 Data Analysis
The software IDLeQuery developed to address the issue of well identification has
been expanded to include automated binning and error checking in the commercial
release called HyperView.
4.5.5 Carryover
The tubing for sample delivery results in carryover of particles and fluids. A thin film
of sample fluid is left behind, which amounts to about 10%of the total sample volume.
The carryover of active compounds can contribute to the behavior of the next sample if
the contents of the well are not at a stable end point or a binding reaction is reversible.
The carryover of particles in bead-based assays is typically less than 1%, but can vary
according to the adherence characteristics and size of cells.
4.5.6 Beads
Bead-based molecular assays are now commonplace in flow cytometry. Our experi-
ence suggests that the approach is generalizable tomost molecular assemblies, as long
as one molecule is stably attached to the bead and the binding of the other molecule
results in a fluorescent signal. For screening large libraries of compounds, assay
conditions and costs need to be evaluated in conjunction with reagent manufacturers
to ensure access to required reagent quantities at appropriate costs. In addition to
targets on beads described here, GPCR [59], kinase [60], and metalloprotease
targets [61, 62] have been addressed outside the MLPCN.
4.5.7 Protein-Protein Interaction Targets
Protein-protein interaction (PPI) targets have so far met with variable success with the
Molecular Libraries Small Molecule Repository. Two targets described here, Bcl-2
family and RGS family multiplexes, have both identified reactive molecules, whereas
the MEKK and proteasome targets identified significant numbers of fluorescent
compounds.
4.5.8 Chemical Probe Development Plan and Secondary Assays
The MLPCN production phase has installed a rigorous process of Chemical Probe
Development Plan (CPDP) in which timelines and responsibilities for primary and
secondary assays are assigned. Independent labs undertaking screening campaigns
should have detailed plans in advance for verifying that compound activities identified
in HTS represent the desired biological effect rather than a physical or chemical
artifact.
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