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2009 ; 486
(ä): 151-65
Nephropedia Template TP
Edwards BS
; Young SM
; Ivnitsky-Steele I
; Ye RD
; Prossnitz ER
; Sklar LA
Methods Mol Biol
2009[]; 486
(ä): 151-65
PMID19347622
show ga
The HyperCyt high-throughput (HT) flow cytometry sampling platform uses a
peristaltic pump, in combination with an autosampler, and a novel approach to
data collection, to circumvent time-delay bottlenecks of conventional flow
cytometry. This approach also dramatically reduces the amount of sample aspirated
for each analysis, typically requiring ~2 microL per sample while making
quantitative fluorescence measurements of 40 or more samples per minute with
thousands to tens of thousands of cells in each sample. Here, we describe a
simple robust screening assay that exploits the high-content measurement
capabilities of the flow cytometer to simicroltaneously probe the binding of test
compounds to two different receptors in a common assay volume, a duplex assay
format. The ability of the flow cytometer to distinguish cell-bound from free
fluorophore is also exploited to eliminate wash steps during assay setup. HT flow
cytometry with this assay has allowed efficient screening of tens of thousands of
small molecules from the NIH Small-Molecule Repository to identify selective
ligands for two related G-protein-coupled receptors, the formylpeptide receptor
and formylpeptide receptor-like 1.
free
free
free
