Biomedical Engineering Reference
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factors. This assay was identified as a result of our outreach effort and was developed
collaboratively. The RNAIII promoter, activated in the late stages of the pathway, is
fused to a GFP expression vector. An active test compound is one that prevents GFP
expression in the presence of a pathway-activating peptide. Secondary assay evidence
suggests that the two most active compounds from different families act at different
points in bacterial quorum sensing pathways as described in the probe reports.
Lessons Learned : HTS for suspension bacterial assays is readily compatible with
flow cytometry when the cells express adequate levels of fluorescent GFP marker.
4.4.7 Activators of Prostate Cell Differentiation Target:
1X01MH078937-01 (Adherent Cell Phenotypic Assay)
The lymph node cancer of the prostate (LNCaP) human prostate cell differentiation
model was used to determine the ability of test compounds to induce a phenotype
with increased side scatter caused by intracellular granule accumulation in prostate
cells in the assay contributed by T. Thompson. Significant optimization was
required for this assay due to the adherent nature of the cells, the passage-dependent
alteration of the responsiveness of the androgen-dependent target cell line (LNCaP),
and the reformatting from 96 to 384-wells that reduced the cell number in the assay
relative to the assay volume. Two new secondary assays, one involving an
androgenic activity assay measured by GFP expression in the flow cytometer [47]
and the other involving androgen-independent granularity increases in PC3 cells,
have been developed in collaboration with the target provider. Hits from the screens
are capable of increasing prostate cell granularity and inducing cell death. Probe
criteria have been met for a phenotypic assay, yielding micromolar activities in
LNCaP and PC3 phenotypic assays [48]. The target provider is following up on
identifying the mechanism of action of
increased granularity, presumably
autophagy.
Lessons Learned : Light scatter responses are readily adapted to HT flow cytometry.
After the screen was conducted, further studies have helped to elucidate the granule
accumulation mechanism as autophagy. The PC3 cells were less adherent than the
LNCaP cells and were readily resuspended for flow cytometry.
4.4.8 Multiplexed High-Throughput Screen for Small-Molecule Regulators
of Bcl-2 Family Protein Interactions: NIH 1X01MH079850-01 (Bead-Based
Multiplexes)
Apoptosis is regulated in part by the balance of antiapoptotic and proapoptotic Bcl-2
family members. In humans, six genes have been identified that encode antiapoptotic
proteins characterized by the presence of conserved motifs designated as three Bcl-2
homology (BH) regions, BH1, BH2, and BH3. These domains form a hydrophobic
cleft in tertiary structure. Proapoptotic family members contain BH3 regions that
form an amphipathic helix, and these helices bind in the clefts of the antiapoptotic
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