High-Throughput Microfluidic Mixing and Multiparametric Cell Sorting for Bioactive Compound Screening

Abstract

, 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 ® 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~10,000 cells analyzed per reaction. Cell Ca 2+ 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. (Journal of Biomolecular Screening 2004:103-111)

Integration of Virtual Screening with High-Throughput Flow Cytometry to Identify Novel Small Molecule Formylpeptide Receptor Antagonists

“…As described previously (Young et al, 2004), 10 7 cells are resuspended in 10 ml of warm medium containing 200 nM Fluo4 acetoxymethyl ester (Invitrogen) and incubated at 37°C for 30 min, with mixing every 10 min. After incubation, cells are washed twice by centrifugation and resuspended in complete HH buffer [110 mM NaCl, 30 mM HEPES, 10 mM KCL, 1 mM MgCl 2 , 10 mM glucose, and 0.1% (v/v) human serum albumin, and 1.5 mM CalCl 2 ].…”

Bioassay‐directed fractionation for discovery of bioactive neutral lipids guided by relative mass defect filtering and multiplexed collision‐induced dissociation

“…Once a receptor ligand is identified, efforts to accelerate compound discovery often generate large libraries of compounds with structural similarity to known active substances, followed by activity screening 3, 4. Complementary efforts have focused on reducing discovery time by improving technologies for bioactivity assays and compound fractionation, including development of high‐throughput microfluidic assays,5 two‐dimensional high‐performance liquid chromatography (2‐D HPLC), and microdialysis‐HPLC methods 6…”

High‐throughput screening and characterization of clones selected from phage display libraries

“…Sets of microspheres with nearly 100 different optical codes are commercially available (for example, Luminex xMAP beads) as are flow cytometers with microplate autosamplers capable of delivering several samples per minute (for example Luminex100 or Becton Dickinson FACSArray), making it possible to screen more than 20,000 clones per hour with commercially available reagents and instruments. If coupled to custom-developed high-throughput sample handling for flow cytometry capable of delivery rates of a sample per second (17)(18)(19), the analysis rate extends into hundreds of thousands per hour. Thus, this approach eliminates any throughput bottleneck associated with screening or characterizing individual clones.…”