DNA Fragmentation Assays for Apoptosis
One of the most easily measured features of apoptotic cells is the break-up of genomic DNA by cellular nucleases. 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. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a gold-standard assay for detecting DNA fragmentation by labeling the terminal end of nucleic acids.
Apo-Direct Apoptosis Detection Kit and Apo-BrdU Apoptosis Detection Kit detect apoptotic cells by flow cytometry. Each kit contains all the reagents required for measuring apoptosis in your cells, including:
- Positive and negative control cells for assessing reagent performance.
- All necessary buffers for processing individual steps in the assay.
- Terminal deoxynucleotidyl transferase enzyme (TdT).
- FITC-dUTP (Apo-Direct) or Br-dUTP plus FITC-labeled Anti-BrdU (Apo-BrdU).
- Propidium Iodide/RNase A solution for counterstaining the total DNA.
Late-Stage Apoptotic Cells
ApoSTAIN Anti-ssDNA allows you to specifically stain condensed chromatin of apoptotic cells, a hallmark of apoptosis. Unlike other apoptosis assays and kits, this unique reagent provides specificity based on the high sensitivity of DNA in the condensed chromatin of apoptotic cells to thermal denaturation. Stain cells and/or tissue sections with ApoSTAIN following heat treatment, which induces DNA denaturation in situ only in apoptotic nuclei. In the presence of formamide, only apoptotic (and not necrotic) nuclei DNA becomes denatured and detectable. Thus, ApoStain is a specific and sensitive method for the detection of apoptotic cells.
- Versatile – Suitable to detect apoptotic cells in various species including mouse, rat and human cells.
- Specific – Does not stain non-apoptotic or necrotic cells.
- Flexible – Use for flow cytometry and immunohistochemical staining of formalin-fixed paraffin embedded tissue sections.
ApoSTAIN Performance
ICC Staining of Staurosporine-treated MB-MDA-468 Cells
Immunocytochemistry of staurosporine-treated MB-MDA-468 cells following formamide incubation and using ApoSTAIN Anti-ssDNA followed by Anti-Mouse IgM FITC. Paired images of the same field show nuclei counterstained with DAPI (left) and ApoSTAIN-positive nuclei (right). Detection of condensed chromatin in three apoptotic cells is clearly visible (right).
