Image - Barts and The London logo and link to home page Image - divider Image - divider
 
  Institute of Cell and Molecular Science
Flow Cytometry Core Facility equipment, images used with permission by BD
  Flow Cytometry Core Facility
  link Home link Instruments available link Flow cytometry link Uses of flow cytometry link Flow sorting link Links link Contacts
 

Caspase activity

 

There are a number of caspases in mammalian cells that have been shown to be involved in the early stages of apoptosis, e.g. Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9 and Caspase 10. The functions of these enzymes are not yet entirely clear, but it appears that after an initial signal to the cell to undergo apoptosis, they may be responsible for the activation, amplification and execution of the apoptotic cascade. Because of the central importance of the caspases in apoptosis, their detection by flow cytometry has become widespread. The activity of enzymes implicated in apoptosis may be detected in three ways:

  • By detecting the active form of the enzyme using a specific antibody. Various forms of cell death e.g. Oncosis, Apoptosis & Necroptosis display various levels of Caspase-3, see figure
  • ER stress only displays background levels of Caspase-3 activiyt, see figure
  • By using a fluorochrome labelled inhibitor peptide that binds to the active site of the caspase or FLICA - Fluorescent Labelled Inhibitor Caspase. Invitrogen kit V35117 includes FAM-VAD-FMK poly caspases, Propidium Iodide and Hoechst 33342 for live and dead cell imaging. The Hoechst supplied in this kit can be used for live cell cell cycle analysis to determine if caspase activation is cell cycle dependent
  • By using a non-fluorescent substrate for the enzyme which yields a fluorescent product if the enzyme is active - eg PhiPhiLux® (http://phiphilux.com/phi1.html [new window])
 
Top
 

Caspase-3 antibody

Capsase-3 activity in Oncosis, Apoptosis & Necroptosis

ER Stress

Invitrogen kit V35117

Cell cycle analysis of caspase activation

by Gary Warnes. © Queen Mary, University of London 2007
Institute of Cell and Molecular Science, The Blizard Building, Barts and The London School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, UK Tel: +44 (0)20 7882 2483, Fax: +44 (0)20 7882 2200