Invitrogen™ Apo-BrdU Apoptosis Detection Kit
On demandInvitrogen™ Apo-BrdU Apoptosis Detection Kit
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 5 mL positive and 5 mL negative control cell suspensions of
approximately 1x106 cells per mL in 70% (v/v) ethanol.
There are no specifications
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