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Cat. No.: LRAKW8-100 (for 100T)

Cat. No.: LRAKW8-500 (for 500T)

Description

LDH Release Assay Kit with WST-8, also known as the LDH Assay Kit with WST-8, LDH Cytotoxicity Assay Kit with WST-8, or LDH Activity Assay Kit with WST-8, is a colorimetric assay kit based on WST-8 that rapidly and sensitively detects lactate dehydrogenase (LDH) activity released due to cell membrane integrity loss, such as in cell necrosis. It can also be used to detect LDH activity in other samples.

This kit is used for cytotoxicity testing based on LDH release and for routine LDH activity detection.

LDH is an enzyme present in most mammalian live cells and is found abundantly in tissues like the heart, skeletal muscle, and kidneys. It is a crucial marker in clinical myocardial enzyme profile tests and assists in diagnosing myocardial diseases. LDH catalyzes the conversion of lactate to pyruvate, accompanied by the conversion of NAD+ to NADH. It is considered a biomarker for cell necrosis, tissue injury, and related diseases because it is released during tissue damage due to cell membrane disruption, leading to the release of intracellular LDH.

Cell death includes apoptosis, necrosis, and pyroptosis. Programmed cell death (PCD) refers to regulated cell death, whereas unregulated cell death is termed necrosis. Necrosis, secondary necrosis from apoptosis, and pyroptosis disrupt cell membrane structure, releasing intracellular enzymes like LDH. In cultured cells, LDH is released into the culture medium, while in vivo it is released into the tissue microenvironment and possibly the bloodstream. Quantitative analysis of LDH activity released from membrane-compromised cells allows for the assessment of cytotoxicity. LDH release is a key indicator of cell membrane integrity, widely used for detecting cell necrosis and membrane damage, and is considered a safe alternative to radioactive 51Cr-labeled cell membrane integrity assays.

LDH activity can be measured by various colorimetric methods such as DNP, INT, and WST-8. The DNP method, based on the reaction of pyruvate (produced by LDH activity) with 2,4-dinitrophenylhydrazine, is less commonly used due to its complexity. The INT method involves the reduction of NAD+ to NADH, which reacts with INT (2-p-iodophenyl-3-nitrophenyl tetrazolium chloride) to produce a formazan dye. The WST-8 method, employed in this kit, generally offers higher sensitivity, greater absorbance change, and more accurate results compared to the INT method.

This kit can detect both D-LDH and L-LDH, as well as their mixtures.

Components

• LDH Release Assay Buffer
• Chromogen Solution
• Lactate Dehydrogenase (2.5U/ml)
• Stop Solution

Principle

Under the action of LDH, NAD+ is reduced to NADH. NADH then reacts with WST-8 to generate NAD+ and a water-soluble formazan dye. This reaction produces an absorption peak at 450nm, allowing for the quantification of LDH activity through colorimetric analysis. The absorbance at 450nm is linearly correlated with LDH activity.

Feature

High Sensitivity and Wide Linear Range: This kit offers high sensitivity and a broad linear range. With a sample volume of 100μl, it can detect LDH activity as low as 0.15mU/ml, maintaining a good linear relationship within the activity range of 0.15-50mU/ml.

Storage

• Store at -20ºC for up to one year.
• Chromogen Solution should be stored protected from light.
• After thawing, the kit can be stored at 4ºC for short periods, effective for 2-3 days.

Precautions

• Freezing can inactivate some of the LDH in samples. Samples can be stored at 4ºC for 2-3 days. It is recommended to measure the samples on the same day they are prepared.
• Ensure the LDH Release Assay Buffer is fully thawed and equilibrated to room temperature before use to avoid affecting the results. Other solutions should be kept on ice during use.
• When detecting LDH in cell culture media, the presence of serum, which contains LDH, can increase background readings. Set up control wells without cells but with the same volume of culture media to eliminate the background. The higher the serum content, the higher the background value. If the experiment is not significantly affected, use inactivated serum to reduce the background substantially, or use serum-free or low-serum media during the experiment to effectively lower the baseline LDH activity from serum.
• Factors such as overgrowth of cells, high cell density, high centrifugation speed, and large temperature differences inside and outside the incubator can increase LDH release from cells. The LDH content varies among different cell types.
• Without adding stop solution, the absorbance will gradually increase over time. After adding stop solution, the coloration can be stably preserved for 48 hours.
• This product is intended for scientific research use by professionals only. It is not for clinical diagnosis or treatment, nor for use in food or medicine. Do not store it in common household environments.
• For your safety and health, please wear a lab coat and disposable gloves when handling.

Only for research and not intended for treatment of humans or animals

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