Flow Cytometry Product Notes: Test Sizes: To accommodate multicolor flow cytometry, eBioscience is in the process of reducing test size volumes from 20 µl to 5 µl. Please check your antibody vial for the recommended test size. Fluorochrome Replacements: eBioscience is in the process of replacing all Alexa Fluor® 647 conjugated products with eFluor® 660 conjugated products.
Description
The APO-BRDU™ Kit is a 2-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 transferase enzyme (TdT), bromodeoxyuridine triphosphate (Br-dUTP), and fluorescein labeled anti-BrdU antibody for labeling DNA breaks 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 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. This greater incorporation gives rise to a brighter flow cytometry signal when the Br-dUTP sites are identified by a fluorescein labeled anti-BrdU monoclonal antibody. Non-apoptotic cells do not incorporate significant amounts of the Br-dUTP due to the lack of exposed 3'-hydroxyl DNA ends.
Sufficient reagents are provided to process a total of 60 cell suspensions including 5ml positive and 5ml negative control cell suspensions of approximately 1x106 cells per ml in 70% (v/v) ethanol.
Protocol
To view protocol, please download the TDS by clicking the PDF button above.
