Fluorochrome-labeled inhibitors of caspases: convenient in vitro and in vivo markers of apoptotic cells for cytometric analysis.
Activation of caspases is a hallmark of apoptosis. Several methods, therefore, were developed to identify and count the frequency of apoptotic cells based on the detection of caspases activation. The method described in this chapter is based on the use of fluorochrome-labeled inhibitors of caspases (FLICA) applicable to fluorescence microscopy, and flow- and image-cytometry. Cell-permeant FLICA reagents tagged with carboxyfluorescein or sulforhodamine when applied to live cells in vitro or in vivo, exclusively label cells that are undergoing apoptosis. The FLICA labeling methodology is simple, rapid, robust, and can be combined with other markers of cell death for multiplexed analysis. Examples are presented on FLICA use in combination with a vital stain (propidium iodide), detection of the loss of mitochondrial electrochemical potential, and exposure of phosphatidylserine on the outer surface of plasma cell membrane using Annexin V fluorochrome conjugates. Following cell fixation and stoichiometric staining of cellular DNA, FLICA binding can be correlated with DNA ploidy, cell cycle phase, DNA fragmentation, and other apoptotic events whose detection requires cell permeabilization. The "time window" for the detection of apoptosis with FLICA is wider compared to that with the Annexin V binding, making FLICA a preferable marker for the detection of early phase apoptosis and more accurate for quantification of apoptotic cells.
Department of Pathology, New York Medical College, Brander Cancer Research Institute, Valhalla, NY, USA.
Zbigniew
Z
Darzynkiewicz
Piotr
P
Pozarowski
Brian W
BW
Lee
Gary L
GL
Johnson
D017209:Apoptosis / Q000187:drug effects*; D015415:Biomarkers / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D047569:Laser Scanning Cytometry; D011480:Protease Inhibitors / Q000494:pharmacology*; D013194:Staining and Labeling
D015415:Biomarkers; D061945:Caspase Inhibitors; D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D020169:Caspases