The Phoenix Flow Systems, Inc. APO-DIRECT 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), Fluorescein-deoxyuridine triphosphate 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.
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 directly conjugated fluorescein- deoxyuridine triphosphate nucleotides (FITC-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-DIRECT Kit. Non-apoptotic cells do not incorporate significant amounts of the FITC-dUTP owing to the lack of exposed 3'-hydroxyl DNA ends. The deoxynucleotide triphosphates complexed to a larger ligand like fluorescein, (biotin or digoxigenin are others) are used in this kit. These larger ligands do not give as strong a fluorescence signal as our APO-BRDU kit. This generalization applies to our APO-DIRECT kit as well as our competition's kits, ie, Oncor and Boehringer Mannheim. We strongly recommend if your experimental protocol does not require the FITC dUTP methodology, that you look at the APO-BRDU kit. Not only is the APO-BRDU kit more sensitive, it costs less per reaction. Both kits are priced the same, but the APO-DIRECT kit contains 50 reactions and the APO-BRDU kit contains 60 reactions.(1).
Link to Cell Fixation procedure
