Development of a Human Whole Blood Screening Platform to Monitor JAK/STAT Signaling Using High-Throughput Flow Cytometry

J Biomol Screen. 2016 Sep;21(8):866-74. doi: 10.1177/1087057116645095. Epub 2016 May 3.


Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the automation technologies and process optimizations developed here overcame sample integrity, handling, and multiparametric data analysis bottlenecks without affecting assay performance. Together these efforts dramatically increased sample throughput compared to conventional manual flow cytometric approaches and enabled development of novel JAK/STAT inhibitors.

Keywords: JAK/STAT signaling; automation; flow cytometry; high throughput; human whole blood; miniaturization.

MeSH terms

  • Flow Cytometry / methods*
  • High-Throughput Screening Assays / methods*
  • Humans
  • Janus Kinases / antagonists & inhibitors
  • Janus Kinases / genetics
  • Phosphorylation
  • Protein Kinase Inhibitors / chemistry
  • Protein Kinase Inhibitors / isolation & purification*
  • STAT Transcription Factors / antagonists & inhibitors
  • STAT Transcription Factors / genetics
  • Signal Transduction / drug effects*
  • Small Molecule Libraries / chemistry


  • Protein Kinase Inhibitors
  • STAT Transcription Factors
  • Small Molecule Libraries
  • Janus Kinases