Description
Key facts
Detection method:Colorimetric,
Sample types:Adherent cells, Suspension cells,
Assay type:Enzyme activity (quantitative),
Assay time:1h,
Assay Platform:Microplate reader
Product details:
LDH Assay Kit (Cytotoxicity) ab65393 uses WST for the fast and sensitive detection of LDH released from damaged cells.
The LDH assay, also known as LDH release assay, is a cell death / cytotoxicity assay used to assess the level of plasma membrane damage in a cell population. Lactate dehydrogenase (LDH) is a stable enzyme, present in all cell types, which is rapidly released into the cell culture medium upon damage of the plasma membrane. LDH is the most widely used marker used to run a cytotoxicity assay.
How the assay works
The LDH assay protocol is based on an enzymatic coupling reaction: LDH released from the cell oxidizes lactate to generate NADH, which then reacts with WST to generate a yellow color. The intensity of the generated color correlates directly with the number of lyzed cells.
Only 10μl of culture medium is required for the assay, and thus background from serum and culture medium is significantly reduced. Cells can be cultured in regular 10% serum containing medium; no reducing serum or special medium is required for the assay.
This LDH assay kit uses a chemical method to couple the NADH reaction with WST, which is more tolerant of variability in the sample than the diaphorase-based coupling method used in kits available from other vendors. The chemical method means that the active ingredients of the kit, excluding the LDH positive control, do not contain enzymes, meaning they are more stable for practical use in the lab in repeated experiments.
In addition, the WST used in the kit is highly stable, soluble in solution and unlikely to precipitate in solution, unlike the INT used in kits available from other vendors. This means that the reaction can be read multiple times over a long time-course. The reaction can also be stopped at any time point.
LDH assay protocol summary
- Transfer 10μl culture medium into new plate
- Add LDH reaction mix and incubate for 30 min at room temp
- Analyze with microplate reader
LDH activity can be easily quantified by spectrophotometer or plate reader at OD450nm.
Related LDH assay products
If you would like to use a fluorometric assay, please refer to LDH-Cytotoxicity Assay Kit (Fluorometric)
ab197004
This kit is more sensitive than the colorimetric LDH Cytoxicity Assay Kit
ab65391
To measure LDH activity in sample types such as serum, plasma, and cell lysates, we recommend LDH assay kit
ab102526
Other notes
This product is manufactured by BioVision, an Abcam company and was previously called K313 LDH-Cytotoxicity Colorimetric Assay Kit II. K313-500 is the same size as the 500 test size of ab65393.
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REACH authorisation
Abcam has not and does not intend to apply for the REACH Authorisation of customers' uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information-Please refer to protocols
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Lactate dehydrogenase (LDH) also known as LD or LDH1 is an enzyme that plays an important role in the conversion between lactate and pyruvate. It catalyzes the oxidation of L-lactate to pyruvate while simultaneously reducing NAD+ to NADH. LDH is approximately 140 kDa in size and consists of tetrameric structures formed by different combinations of its subunits. Expression of LDH occurs in many tissues with significant presence in muscles liver kidneys and the heart.
Biological function summary
The enzyme works efficiently to help cells convert glucose into energy under conditions lacking oxygen such as intense exercise or hypoxia. LDH does not act alone; it functions as part of a larger metabolic network where it works with other enzymes and proteins to maintain cellular energy balance. The enzyme helps in maintaining the redox balance within the cell by regulating levels of NAD+ and NADH. Due to its importance in metabolism LDH is often measured in laboratories using lactate dehydrogenase assays including LDH release assays and LDH activity assays.
Pathways
LDH is an integral part of the glycolysis and gluconeogenesis pathways. It facilitates the conversion of pyruvate to lactate especially during anaerobic glycolysis enabling the continual production of ATP in oxygen-poor environments. LDH is associated with other metabolic proteins like pyruvate kinase and hexokinase each playing their own roles in energy production processes. Understanding these interactions is important in research that involves energy metabolism.
LDH levels can indicate tissue damage or disease progression. Elevated LDH levels are often associated with conditions such as myocardial infarction and various types of cancers. For example in cancer LDH levels increase due to high anaerobic glycolysis rates commonly observed in tumor cells. LDH also relates to diagnostic markers in myocardial infarctions where it increases in response to cardiac tissue damage. LDH assays are frequently used as part of diagnostic protocols to assess the severity or presence of such conditions.
