A Homogeneous Assay to Measure Live and Dead Cells in the Same Sample by Detecting Different Protease Markers

Anal Biochem. 2007 Jul 15;366(2):197-206. doi: 10.1016/j.ab.2007.04.007. Epub 2007 Apr 12.


A method to simultaneously determine the relative numbers of live and dead cells in culture by introducing a combination of two fluorogenic substrates or a fluorogenic and a luminogenic protease substrate into the sample is described. The method is based on detection of differential ubiquitous proteolytic activities associated with intact viable cells and cells that have lost membrane integrity. A cell-permeable peptide aminofluorocoumarin substrate detects protease activity restricted to intact viable cells. Upon cell death, the viable cell protease marker becomes inactive. An impermeable peptide rhodamine 110 (or aminoluciferin) conjugated substrate detects protease activity from nonviable cells that have lost membrane integrity. The multiplex assay can detect 200 dead cells in a population of 10,000 viable cells. The protease substrate reagents do not damage viable cells over the course of the assay, thus the method can be multiplexed further with other assays in a homogeneous format. Ratiometric measurement of viable and dead cells in the same sample provides an internal control that can be used to normalize data from other cell-based assays.

MeSH terms

  • Apoptosis / drug effects
  • Biomarkers / analysis
  • Biomarkers / chemistry
  • Biomarkers / metabolism*
  • Cell Line
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Coumarins / chemistry
  • Coumarins / metabolism
  • Dose-Response Relationship, Drug
  • Fluorescent Dyes / chemistry*
  • HCT116 Cells
  • HL-60 Cells
  • HeLa Cells
  • Humans
  • Ionomycin / pharmacology
  • Jurkat Cells
  • Peptide Hydrolases / analysis
  • Peptide Hydrolases / chemistry
  • Peptide Hydrolases / metabolism*
  • Rhodamines / chemistry
  • Rhodamines / metabolism
  • Staurosporine / pharmacology
  • U937 Cells


  • Biomarkers
  • Coumarins
  • Fluorescent Dyes
  • Rhodamines
  • Ionomycin
  • Peptide Hydrolases
  • Staurosporine