SIMPLE AND RAPID METHOD FOR DETECTION OF APOPTOTIC CELLS BY FLOW-CYTOMETRY

We have developed a simple and rapid method for detection of apoptotic cells by flow cytometry-a modified nick end-labeling method. DNA fragments in situ associated with apoptosis are detected by the nick-end labeling method (TUNEL method) in our new method. This method is well suited for easy identification of cell types and for in situ measurement of minute quantities of apoptotic cells. We present a modified method for nick-end labeling by flow cytometry (NELF) to detect apoptosis of both cultured cells and blood cells and also to analyze DNA fragments quantitatively and at the single-cell level. We modified the NELF method to simplify procedures and to save time. The protocol consists of 1) fixation with cold acetone, 2) transfer of biotinylated nucleotide to the 3'-OH end with terminal deoxynucleotidyl transferase, and 3) incubation with streptoavidin-conjugated phycoerythrin to biotinylated nucleotide. This method may be useful in two-color analysis with a wide variety of antibodies.