期刊
JOURNAL OF BIOMOLECULAR SCREENING
卷 9, 期 2, 页码 103-111出版社
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057103262335
关键词
drug discovery; flow cytometry; automation; cell sorting; sample handling
资金
- NCI NIH HHS [R24-CA88339] Funding Source: Medline
- NIAID NIH HHS [AI48517] Funding Source: Medline
- NIBIB NIH HHS [EB00264] Funding Source: Medline
- NIGMS NIH HHS [R24-GM60799] Funding Source: Medline
Hypei-Cyt(R), an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt(R) configuration, air bubble-separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with similar to 10,000 cells analyzed per reaction. Cell Ca2+ responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.
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