Analysis of apoptosis by cytometry using TUNEL assay

Abstract

Activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. DNA fragmentation revealed by the presence of a multitude of DNA strand breaks, therefore, is considered to be the gold standard for identification apoptotic cells. Several variants of the methodology that is based on fluorochrome-labeling of 3'-OH termini of DNA strand breaks in situ with the use of exogenous terminal deoxynucleotidyl transferase (TdT), commonly defined as the TUNEL assay, have been developed by us. This Chapter describes the variant based on strand breaks labeling with Br-dUTP that is subsequently detected immunocytochemically with Br-dUAb. Compared with other TUNEL variants the Br-dU-labeling assay offers the greatest sensitivity in detecting DNA breaks. Described also are modifications of the protocol that allow one to use other than Br-dUTP fluorochrome-tagged deoxynucleotides to label DNA breaks. Concurrent staining of DNA with propidium or 4',6-diamidino-2-phenylindole (DAPI) and multiparameter analysis of cells by flow- or laser scanning cytometry enables one to correlate induction of apoptosis with the cell cycle phase.

Fig 1

Schematic illustration of DNA strand breaks labeling with Br-dUTP utilizing exogenous terminal deoxynucleotidyl transferase (TdT) as described in this chapter.

Fig 2. Scatterplots illustrating the detection of DNA strand breaks in apoptotic cells by TUNEL assay

HL-60 cells were untreated (Ctrl) or treated with 200 nM DNA topoisomerase 1 inhibitor camptothecin (CPT) for 3 h , fixed and processed according to the protocol described in this chapter. Cellular DNA was stained with PI while DNA strand breaks were labeled with BrdUTP followed by the FITC-conjugated BrdU Ab. Based on differenced in DNA content one can identify cells in G₁ vs S vs G₂M phases of the cell cycle as shown in the left panel (separated by the dashed vertical boundaries). Apoptotic cells (Ap) are characterized by very high frequency of DNA strand breaks (note exponential scale of Y coordinate). It is quite evident that CPT induced apoptosis preferentially of S-phase cells.