Description of Kit

The Phoenix Flow Systems, Inc. APO-BRDU Kit is a two color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry. The kit contains the instructions and reagents required for measuring apoptosis in cells including; positive and negative control cells for assessing reagent performance; washing, reaction and rinsing buffers for processing individual steps in the assay; terminal deoxynucleotidyl tranferase enzyme (TdT), bromodeoxyuridine triphosphate (Br-dUTP), and fluorescein labeled antiBrdU antibody for labeling DNA breaks and propidium iodide/RNase A solution for counter staining the total DNA. This kit also contains an evaluation copy of APO-SOFT software to illustrate the power of dual parameter subtraction for the analysis of apoptotic data.

How the Kit works (A Tubel NOT Tunel Assay)

One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of “DNA laddering” when the DNA is analyzed by agarose gel electrophoresis. The DNA of non-apoptotic cells which remains largely intact does not display this “laddering” on agarose gels during electrophoresis. The large number of DNA fragments appearing in apoptotic cells results in a multitude of 3’-hydroxyl termini in the DNA. This property can be used to identify apoptotic cells by labeling the 3’-hydroxyl ends with bromolated deoxyuridine triphosphate nucleotides (Br-dUTP). The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes a template independent addition of deoxyribonucleoside triphosphates to the 3’-hydroxyl ends of double- or single-stranded DNA with either blunt, recessed or overhanging ends. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the APO-BRDU Kit. Recent evidence has demonstrated that Br-dUTP is more readily incorporated into the genome of apoptotic cells than are the deoxynucleotide triphosphates complexed to larger ligands like fluorescein, biotin or digoxigenin (1). This greater incorporation gives rise to a brighter flow cytometry signal when the Br-dUTP sites are identified by a fluorescein labeled antiBrdU monoclonal antibody. Non-apoptotic cells do not incorporate significant amounts of the Br-dUTP owing to the lack of exposed 3’-hydroxyl DNA ends.

The protocol