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Cell Death Detection ELISA^PLU/Cell Death Detection ELISA PLUS
Application
The Cell Death Detection ELISAPLUS photometric enzyme immunoassay is used for the quantitative in vitro determination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) after induced cell death.
The kit contains a stop solution which allows users to terminate the substrate reaction and run the assay under defined conditions, making it suitable for use in high-throughput applications.
Product Description
Assay time:
3 - 4 hours
Negative control:
Depending on cell culture conditions, each exponentially growing permanent cell culture contains a certain amount of dead cells (typically approximately 3 - 8%). In the immunoassay, these inherent dead cells in the untreated sample (without a cell-death-inducing agent) will cause a certain absorbance value (negative control).
Positive control:
A DNA-histone complex serves as a positive control.
Sample material:
Cytoplasmic fractions (lysates) of cell lines, cells ex vivo, cell culture supernatants, and serum or plasma
Sensitivity:
The exact detection limit of dying/dead cells in a particular sample strongly depends on the kinetics of cell death, the cytotoxic agent used, and the amount of affected cells in the total cell population. Using U937/camptothecin (CAM) as a cellular model system for cell death, the immunoassay allows the specific detection of mono- and oligonucleosomes in the cytoplasmic fraction of 125 cell equivalents/well.
Specificity:
Anti-histone reacts with the histones H1, H2A, H2B, H3, and H4 of various species (e.g., human, mouse, rat, hamster, cow, opossum, Xenopus). Anti-DNA binds to single- and double-stranded DNA. Therefore, the ELISA allows the detection of mono- and oligonucleosomes from various species, and may be applied to measure apoptotic cell death in many different cell systems.
Contents
Anti-histone-biotin (clone H11-4)
Anti-DNA-POD (clone MCA-33)
Positive Control
Incubation Buffer
Lysis Buffer
Substrate Buffer
ABTS Substrate Tablet
ABTS Stop Solution
Microplate (streptavidin-coated)
Adhesive Cover Foils
Background Information
Two distinct forms of eukaryotic cell death can be classified by morphological and biochemical criteria: necrosis and apoptosis. Necrosis is accompanied by increased ion permeability of the plasma membrane; the cells swell and the plasma membrane ruptures within minutes (osmotic lysis). Apoptosis is characterized by membrane blebbing (zeiosis), condensation of cytoplasm, and the activation of an endogenous endonuclease. This Ca2+- and Mg2+-dependent nuclease cleaves double-stranded DNA at the most accessible internucleosomal linker region, generating mono- and oligonucleosomes. In contrast, the DNA of the nucleosomes is tightly complexed with the core histones H2A, H2B, H3, and H4, and is thus protected from cleavage by the endonuclease. The yielded DNA fragments are discrete multiples of an 180-bp subunit, detected as a “DNA ladder” on agarose gels after extraction and separation of the fragmented DNA. The enrichment of mono- and oligonucleosomes in the cytoplasm of the apoptotic cell is due to the fact that DNA degradation occurs several hours before plasma membrane breakdown. |