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LDH Assay Kit (Cytotoxicity) ab65393 is an addition-only, no-wash assay with just one 30-min incubation at room temperature.

- Complete kit including positive control
- Cited in over 150 publications
- Individual kit components also available for purchase with a minimum order of 20 units. Contact us to discuss your needs.

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Images

Functional Studies - LDH Assay Kit (Cytotoxicity) (AB65393), expandable thumbnail
  • Functional Studies - LDH Assay Kit (Cytotoxicity) (AB65393), expandable thumbnail
  • Functional Studies - LDH Assay Kit (Cytotoxicity) (AB65393), expandable thumbnail
  • Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (AB65393), expandable thumbnail
  • Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (AB65393), expandable thumbnail

Publications

Key facts

Detection method
Colorimetric
Sample types
Adherent cells, Suspension cells
Assay type
Enzyme activity (quantitative)
Assay time
1h

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Target data

Function

Interconverts simultaneously and stereospecifically pyruvate and lactate with concomitant interconversion of NADH and NAD(+).

Alternative names

What's included?

10000 Test
Components
LDH Assay Buffer
20 x 50 mL
LDH Positive Control
20 x 1 Vial
Lysis Buffer II
20 x 5 mL
Stop Solution IV
20 x 5 mL
Substrate Mix
20 x 1 Vial

Recommended products

LDH Assay Kit (Cytotoxicity) ab65393 is an addition-only, no-wash assay with just one 30-min incubation at room temperature.

- Complete kit including positive control
- Cited in over 150 publications
- Individual kit components also available for purchase with a minimum order of 20 units. Contact us to discuss your needs.

Key facts

Detection method
Colorimetric
Sample types
Adherent cells, Suspension cells
Assay type
Enzyme activity (quantitative)
Assay time
1h
Assay Platform
Microplate reader

Storage

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

Notes

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) LDH-Cytotoxicity Assay Kit (Fluorometric) ab197004.

This kit is more sensitive than the colorimetric LDH Cytoxicity Assay Kit LDH Assay Kit (Cytotoxicity) ab65391.

To measure LDH activity in sample types such as serum, plasma, and cell lysates, we recommend LDH assay kit LDH Assay Kit / Lactate Dehydrogenase Assay Kit (Colorimetric) 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.

Explore our curated range of easy-to-use, efficient and fast cell viability assays.

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.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.
Activity summary

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.

Associated diseases and disorders

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.

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10 product images

  • Functional Studies - LDH Assay Kit (Cytotoxicity) (ab65393), expandable thumbnail
    Image from Bukong TN et al., PLoS Pathog 10(10), Fig S6. Doi: 10.1371/journal.ppat.1004424. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Functional Studies - LDH Assay Kit (Cytotoxicity) (ab65393)

    Bafilomycin A1 (BafA1) toxicity was assessed in Human hepatic (Huh7.5) cells after 24 hours at different concentrations (12.5nm, 25nm, 50nm and 100nm) were administered to cells using LDH cytotoxicity assay kit (ab65393). Cytotoxicity was measured by subtracting LDH content in remaining viable cells from total LDH in untreated controls.

  • Functional Studies - LDH Assay Kit (Cytotoxicity) (ab65393), expandable thumbnail

    Functional Studies - LDH Assay Kit (Cytotoxicity) (ab65393)

    Jurkat T cells were cultured in 96-well plate in 100 μl of culture medium. LDH Assay was performed using 10μl of culture medium using the WST probe. Low control (white bar); High control (black bar).

  • Functional Studies - LDH Assay Kit (Cytotoxicity) (ab65393), expandable thumbnail

    Functional Studies - LDH Assay Kit (Cytotoxicity) (ab65393)

    Comparison of WST-1 and INT based LDH assays. 3T3 cells were cultured in a 96-well plate in 100 μl of culture medium. The LDH assay was performed using 10 μl of culture medium using WST-1 (Brown bar) and INT (Green bar) methods. The WST-1 based LDH assay is more stable and sensitive than the INT based method.

  • Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393), expandable thumbnail

    Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393)

    Gaetano et al. used LDH Assay Kit ab65393 to investigate the potential use of nanoparticles based on ACyD8 (ACyD8-NPs) loaded with Idebenone (IDE), a synthetic short-chain analogue of coenzyme Q10, as a potential therapeutic for Parkinson's disease.

    Analysis of cytotoxicity on the neuronal SH-SY5Y cells pretreated with IDE/ACyD8-NPs and exposed to 30 µM H2O2 for 72 h. Concentrations in IDE/ACyD8-NPs refers to the actual loading of IDE within NPs. Unloaded ACyD8-NPs (i) and (ii) were tested at the same ACyD8 concentration used for testing the drug-loaded NPs 20 µM and 40 µM, respectively. Data are presented as mean ± S.D. One-way ANOVA with Tukey’s multiple comparison test: in (a), all data are not statistically different; in (b), * p < 0.05 (H2O2 + IDE/ACyD8-NPs 2:1 40 µM versus H2O2).

    For the cytotoxicity analysis, the cells were pretreated with NPs (conditions 1–4) for 4 h and then exposed to 30 µM H2O2. Cytotoxicity was evaluated with the LDH-Cytotoxicity Assay Kit II (Abcam, Cambridge, UK, ab65393) after 72 h.

  • Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393), expandable thumbnail

    Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393)

    Salama et al. used LDH Assay Kit ab65393 to investigate cytotoxicity when melanoma cell cultures were treated with a potential therapeutic small molecule YO-2.

    The LDH assay kit was used to determine cytotoxicity in B16F10 cells treated with/without YO-2 after 24 h (n = 6).

    B16F10 cells (1 Ч 105 cells/well) were seeded in triplicate in 6-well plates (Thermo Fisher Scientific, Lafayette, CO, USA) and were left for 16 h overnight to attach before the treatment started. We treated cells with/without YO-2, or DMSO/PBS controls for 24 h.

  • Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393), expandable thumbnail

    Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393)

    Chen et al. used LDH Assay Kit ab65393 to investigate cytotoxicity in a placental model of SARS-CoV-2 infection.

    LDH levels in the supernatant of 55F STs during differentiation. n = 3 independent experiments

    Supernatants collected on day 3 after infection (or indicated otherwise) were analysed for LDH levels.LDH was measured using the Abcam LDH cytotoxicity kit II (ab65393) according to the manufacturer’s instructions in undiluted supernatants using an LDH standard curve.

  • Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393), expandable thumbnail

    Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393)

    Lee et al. used LDH Assay Kit ab65393 to investigate the effect of Korean Red Ginseng extract (RGE) on the PD-1/PD-L1 interaction and its antitumor effects using a humanized PD-1/PD-L1-expressing colorectal cancer (CRC) mouse model. The CD8+ T cell-mediated tumor cell killing effect of RGE was evaluated using murine hPD-L1-expressing MC38 cells and tumor-infiltrating hPD-1-expressing CD8+ T cells isolated from hPD-L1 MC38 tumor-bearing hPD-1 mice.

    Cytotoxicity in co-cultured hPD-L1 MC38 cells was detected by an LDH cytotoxicity assay. a co-culture cell system was established using tumor-infiltrating CD8+ T cell as effector cells in conjunction with hPD-L1 MC38 cells as target cells to elucidate the CD8+ T cell-mediated antitumor effect of RGE. The cytotoxicity of hPD-L1 MC38 cells co-cultured with tumor-infiltrated CD8+ T cells was increasingly high in a concentration-dependent manner.

    A lactate dehydrogenase (LDH) assay kit (#ab65393, Abcam, Cambridge, UK) was used to measure the cytotoxic activity on target cells via effector cells according to the manufacturer’s instructions. The absorbance of the formazan products was determined at 450 nm using a SpectraMax i3 microplate reader.

  • Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393), expandable thumbnail

    Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393)

    Snyder et al. used LDH Assay Kit ab65393 to investigate cytoxicity in male and female neurons maintained in next-generation Neurobasal-Plus media and subjected to a mechanical stretch-injury (to model traumatic brain injury).

    Box plots of post-injury LDH levels from 3 independent culture isolations.

  • Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393), expandable thumbnail

    Enzyme activity assay - LDH Assay Kit (Cytotoxicity) (ab65393)

    Mudyanselage et al. used LDH Assay Kit ab65393 to compare the neurotoxicity of OPs, chlorpyrifos-oxon (CPO), and azamethiphos (AZO) and the carbamate pesticide, aldicarb, to undifferentiated versus differentiated SH-SY5Y neuroblastoma cells.

    Toxicity of CPO, AZO, and aldicarb to undifferentiated and differentiated SHSY-5Y cells measured using a LDH assay. Undifferentiated (A) or differentiated (B) SHSY-5Y cells were treated with CPO, AZO, or aldicarb and extracellular LDH production was quantified using an LDH activity assay. Absorbance readings were corrected by subtracting the values of blanks, then the resultant values were normalised to the LDH production from a vehicle control, providing LDH production as a percentage value relative to the vehicle control. Readings were taken from five independent experiments with three replicates for each treatment. Results were analysed using one-way ANOVA with Dunnett’s multiple comparison tests and expressed as means ± standard error of the mean (SEM). For significance, *** p < 0.001, **** p < 0.0001.

    The production of active extracellular LDH in response to pesticide exposures was measured using an LDH assay kit (ab65393, Abcam, Cambridge, UK) according to the manufacturer’s instructions. After agent treatment (1–200 µM, or vehicle control), 50 µL of spent media was removed and LDH activity was measured spectrophotometrically at 450 nm (Multiskan Spectrum, Thermo Electron Corporation, Finland). Assays were performed in triplicates, with blank values from a negative control subtracted from test values. IC50 values were obtained from the concentration-response curves and expressed as means ± standard error of the mean (SEM). Experiments were performed with an n-number of at least five.

  • Biochemical assay - LDH Assay Kit (Cytotoxicity) (ab65393), expandable thumbnail

    Biochemical assay - LDH Assay Kit (Cytotoxicity) (ab65393)

    Diagram showing the principles of the LDH assay method.

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