Invitrogen™ Apo-Direct Apoptosis Detection Kit

Description

• The APO-DIRECT™ Apoptosis Detection Kit includes sufficient reagents to process a total of 50 cell suspensions, including: 5mL positive and 5 mL negative control cell suspensions of approximately 1×10 6 cells per mL in 70% (v/v) ethanol for assessing reagent performance Washing, reaction and rinsing buffers for processing individual steps in the assay Terminal deoxynucleotidyl transferase enzyme (TdT), Fluorescein-deoxyuridine triphosphate and propidium iodide/RNase A solution for counterstaining the total DNA 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 that 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 due to the lack of exposed 3′-hydroxyl DNA ends.