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Cytotoxicity Detection Kit (LDH)
Application
The Cytotoxicity Detection Kit offers a fast, simple way to quantitate cytotoxicity/cytolysis. For example, it can be used to:
Detect and quantify of cell-mediated cytotoxicity.
Determine mediator-induced cytolysis.
Determine the cytotoxic potential of compounds in environmental and medical research, as well as in the food, cosmetic, and pharmaceutical industries.
Determine cell death in bioreactors.
Note: The assay measures LDH activity released from damaged cells. It is performed in 96-well plates.
Benefits
•Flexible, since the assay can determine the degree of plasma membrane damage in many different in vitro cell systems. •Safe, because the assay does not use radioactive isotopes. •Accurate, since assay results strongly correlate to the number of lysed cells. •Sensitive, since the assay can detect even low cell numbers. •Fast, since assay results can be determined on a multi-well ELISA reader, which allows simultaneous processing of many samples. •Convenient, since the assay does not require a prelabeling step.
Product Description
Sample material: Cell-free supernatants obtained from cells cultured in 96-well microplates or batch cultures.
Background Information
Historically, cell death has usually been evaluated by determining plasma-membrane damage.
Widely used methods are based on the differential uptake or exclusion of dyes (e.g., trypan blue, propidium iodide), with stained or unstained cells counted under a microscope. The major disadvantage of these methods is that they cannot readily process a large number of samples.
Another group of assays is based on release of radioactive isotopes (e.g ., [51Cr], [3H]-thymidine) or fluorescence dyes from prelabeled target cells. The disadvantages of these assays are that (i) most of them use radioactive isotopes, (ii) they require prelabeling of the target cells, and (iii) they have high backgrounds, due to the high spontaneous release of the label from the target cells.
A third type of assay is based on measurement of cytoplasmic enzyme activities released by damaged cells. Several enzyme-release assays have been described (e.g., for alkaline and acid phosphatase); however, many of these assays are hampered by (i) the low amount of endogenous enzyme present in many types of cells, and (ii) the elaborate kinetic assays required to quantitate the enzyme activities. In contrast, lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that is present in all cells. LDH is rapidly released into the cell-culture supernatant when the plasma membrane is damaged and is simple to assay.
Contents
0.Catalyst (Diaphorase/NAD+ mixture)0.Dye Solution (INT and sodium lactate)
Principle
Culture supernatant is collected and cells are removed from it. The cell-free supernatant is incubated with the substrate mixture from the kit. LDH activity is determined in a coupled enzymatic reaction; during this reaction, the tetrazolium salt INT is reduced to formazan. This formazan dye is easy to assay, since it is water-soluble and has a broad absorption maximum at approx. 500 nm.
During the assay, LDH enzyme activity in the culture supernatant increases as the number of dead cells (or cells with damaged plasma membranes) increases. The increase in supernatant LDH activity directly correlates to the amount of formazan formed over time.
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