Analysis of apoptosis by laser scanning cytometry

Flow cytometry techniques that are widely used in studies of cell death, an d particularly in the identification of apoptotic cells, generally rely on the measurement of a single characteristic biochemical or molecular attribu te. These methods fail to recognize cell death lacking that attribute, as i n some examples of atypical apoptosis. Since apoptosis was originally defin ed by morphologic criteria, we suggest that for any new cell system the cyt ometry-defined apoptosis be confirmed by morphologic examination. This qual ity assurance measure is now provided by laser scanning cytometry (LSC). LS C measurements of cell fluorescence are precise and highly sensitive, compa rable to flow cytometry (FCM), and can be carried out on cells on slides, p ermitting cell by cell correlation of fluorescence cytometry with visual mi croscopic morphology. In this report wt describe adaptations of various flo w cytometry techniques for detection of apoptosis by laser scanning cytomet ry. We also describe features unique to LSC that are useful in recognizing apoptosis. Hyperchromicity of DNA, reflecting chromatin condensation, is ev idenced by high maximal pixel values for fluorescence of the DNA-bound fluo rochrome. Mitochondrial probes that have been adapted to LSC to measure the drop in mitochondrial transmembrane potential that occurs early in apoptos is include rhodamine 123, 3,3'-dihexiloxadicarbocyanine [DiOC(6)(3)], and t he aggregate dye 5,5',6,6'-tetrachloro-1,1',3,3-tetraethylbenzimidazolcarbo cyanine iodide (JC-1). The changes in plasma membrane phospholipids and tra nsport function, also early in apoptosis, are probed by a combination of th e fluoresceinated annexin V and DNA fluorochromes such as propidium or 7-am inoactino-mycin D. We also review methods of detection of apoptosis based o n analysis of DNA fragmentation and their application to clinical oncology. Visual examination of the presumed apoptotic cells detected by cytometry m akes it possible to discriminate those that are genuine from monocytes/macr ophages that have ingested nuclear fragments via apoptotic bodies. Applicat ions of flow cytometry and laser scanning cytometry in analysis of cell dea th are discussed and their respective advantages and disadvantages compared . (C) 1999 Wiley-Liss, Inc.