Biomedical Engineering Reference
In-Depth Information
cell populations in hematology, immunology, cancer biology, reproductive biology,
marine biology, microbiology, pharmacology, toxicology, and so on. In principle, all
these features are compatible with HyperCyt.
In practice, the flow cytometer measures several 100 particles for an accurate
determination of mean fluorescence for a particle population. Fluorescence signals
are readily detected when there are 10,000 fluorophores per particle. HyperCyt
enables 1
L volumes to be sampled. For liquid handling reasons, we currently require
well volumes
m
1000-10,000 cells or beads per
microliter. Fluorescent molecular assemblies with affinities
10
m
L with particle densities
500 nM can typically
be assayed in homogeneous, no-wash format. Expensive epitope-tagged protein
reagents are conserved when associated with beads that use
<
1 pmol per assay. Less
precious reagents—fluorescent small molecules, peptides, and oligos—can be sup-
plied at concentrations above their K
d
in contrast to other homogeneous binding
methods requiring both sets of reagent concentration near K
d
. These elements are
discussed elsewhere in more detail [30].
<
4.4.1 The UNMCMD Pipeline
The diversity of targets for flow cytometry is illustrated in part by the pipeline of
projects that have entered the UNMCMD through the Molecular Libraries Initiative
(Table 4.3). Pilot-phase projects are described briefly below along with a perspective
on practical lessons learned from each project. Projects initially entered through an
X01 review mechanism without funding for the target provider that was rapidly
replaced by the R03 mechanismwith funding for the target provider. There have been
three review cycles each year, which is often the case for NIH funding initiatives.
Screeningmilestones varied as theMLSMR grew during the course of the pilot phase.
For example, in the first year of the initiative, the library had grown to
65,000
compounds. Our initial screens were carried out against a
10,000 compound
diversity set augmented by a 17,000 compound subset of the library provided by
our collaborator ChemDiv. We were allowed to use this subset by NIH to enhance the
possibility of chemical optimization through synthesis by ChemDiv. Extensive data
for each target are available at PubChem that can be searched by target, center,
compound, and so on. Probes are defined by criteria identified by the target provider,
but typically molecular activity must be
100 nM and cellular activity
1
m
M.
4.4.2 Assay for Formyl Peptide Receptor Ligands: 1R03 MH076381-01
4.4.2.1 Suspension Cell Multiplex
The assay for formyl peptide receptor (FPR) is
a cell-based assay that measures competitive displacement of a FITC fluorescent
receptor ligand by test compounds. The FPR family has been implicated in inflam-
matory responses in leukocytes (FPR) as well as neurological diseases (FPRL1), and
more recently inmetastasis in glioma and ovarian cancer. The assaywas performed as a
duplex, simultaneously measuring test compound effects on two family member
targets, FPR and FPRL1 in two cell lines, one of which had been stained with a red
fluorescent dye [37]. As part of the assay development, a novel fluorescent peptide
Search WWH ::
Custom Search