Lactate dehydrogenase (LDH) assays are used to quantify the release of LDH from damaged cells into the cell culture supernatant.

LDH is a commonly used method for determining cell viability and cell cytotoxicity following cell damage or death, such as by necrosis, apoptosis, and other forms of cell and tissue damage. LDH release assays are applied in various areas of research, such as during pre-clinical drug development, assessing response to treatments ^1,2.

The LDH release assay offers a fast and reliable method for quantifying elevated LDH levels, serving as an effective surrogate marker for cellular damage. It delivers rapid and reproducible results, making it a widely used approach for evaluating the cytotoxic effects of therapeutic compounds³. LDH activity can be detected across a range of biological sources, including plasma, serum, tissues, cells, and culture media. Commercially available LDH assay kits have been successfully adapted for longitudinal monitoring of cell growth and viability in both 2D and 3D culture systems, including xenospheres, patient-derived explants (PDEs), and spheroids⁴.

Biochemical principles of LDH assay

Under normal physiological conditions, glucose metabolism proceeds through glycolysis, where pyruvate is generated and further oxidized via oxidative phosphorylation to produce ATP. However, in anaerobic or hypoxic environments, the activity of lactate dehydrogenase (LDH) increases to sustain energy production through anaerobic glycolysis. LDH is a cytoplasmic oxidoreductase enzyme that catalyzes the reversible conversion of pyruvate to lactate, coupled with the oxidation of NADH to NAD⁺. This reaction involves the transfer of a hydride ion from NADH to the C2 carbon of pyruvate, ensuring the regeneration of NAD⁺ required for continued glycolysis^1,5.

The LDH cytotoxicity assay leverages this biochemical activity to measure cell membrane integrity. When cells undergo damage or lysis, LDH is released into the extracellular environment, where its activity can be detected. The assay relies on a coupled enzymatic reaction in which LDH converts lactate to pyruvate, reducing NAD⁺ to NADH. The resulting NADH subsequently drives the reduction of various chromogenic or fluorogenic substrates, such as tetrazolium salts (INT), resazurin, or luciferase-based reporters, producing measurable colorimetric, fluorescent, or luminescent signals. The intensity of the signal detected in the culture supernatant is proportional to the extent of LDH release and thereby serves as a direct indicator of cell viability or cytotoxicity across a range of tissues, aided by the ubiquitous expression of LDH and multiple isozyme forms^1,3,6.

Types of LDH assays

LDH assays include colorimetric, fluorometric, and bioluminescent types, each differing in sensitivity, detection method, and suitability for assessing cell viability and cytotoxicity.

Colorimetric LDH assays

Colorimetric assays employ the quantitative measurement of cytosolic LDH release via a biochemical marker (dye) to assess the metabolic activity of the cells. The LDH release is monitored by absorbance reading via a spectrophotometer. Colorimetric assays are widely used to determine cell viability and cytotoxicity, as they are easy to use, safe, and economical. LDH present in the culture medium catalyzes the oxidation of lactate to pyruvate coupled with NADH, reducing a tetrazolium salt to formazan, a water-soluble dye^7,8. Several tetrazolium salts are used in colorimetric LDH assays. For example, the LDH cytotoxic assay kit uses iodonitrotetrazolium chloride (INT), which NADH reduces into a red formazan dye. Formazan can be quantified by measuring absorbance at 492 nm and is directly proportional to the amount of cell damage in the culture medium².

Colorimetric assays include the LDH assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, water-soluble tetrazolium-1 (WST-1) assay, water-soluble tetrazolium-8 (WST-8) assay, sulforhodamine B (SRB) assay, neutral red uptake (NRU) assay, and crystal violet assay⁷.

Fluorometric LDH assays

Fluorometric assays offer enhanced sensitivity, broader linearity ranges, and greater throughput compared to colorimetric assays, making them more effective for detecting LDH activity. A commonly used fluorometric method is the resazurin reduction assay, which relies on the conversion of resazurin, a blue, non-fluorescent dye, into resorufin, a pink fluorescent compound. This transformation occurs during a coupled enzymatic reaction, where LDH catalyzes the conversion of lactate to pyruvate, simultaneously reducing nicotinamide adenine dinucleotide (NAD⁺) to NADH. The NADH generated then facilitates the reduction of resazurin to resorufin. The fluorescence intensity of resorufin, measured typically at 560–590 nm, is directly proportional to the metabolic activity of the cells^7,9.

Bioluminescent LDH assays

Bioluminescent assays are the most sensitive and fastest assays that measure LDH activity by generating a luminescent signal. In this assay, the production of NADH accelerates a coupled reaction, where the pro-luciferin substrate is converted to luciferin via a luciferase-catalyzed reaction to generate a luminescent signal, which remains persistent for some time after reagent addition and can be measured through a luminometer. Luminescent assays favor detection in smaller sample sizes, allowing for measurement of small changes in cell viability, and have been widely used in 3D complex cultures^7,10,11.

Mechanism of LDH assay

LDH release and detection

Upon exposure to cytotoxic compounds or conditions, cell membrane integrity is affected, and cells undergo necrosis, apoptosis, or other forms of cell death. In this process, cytoplasmic content leaks into the extracellular medium, including LDH. Given the stability and ability to remain elevated for up to 7 days in the bloodstream and its ubiquitous presence in all cells, LDH serves as a potential marker indicating cell cytotoxicity¹.

Coupled enzyme reactions

The elevated LDH levels mediate the conversion of pyruvate to lactate and NAD⁺ to NADH. Simultaneously, NADH can reduce various substrates such as resazurin, tetrazolium salts, and luminescent molecules, which, when added, stimulate a coupled enzyme reaction to exhibit color or light emission. Quantification of this signal determines the LDH activity and is directly proportional to the number of cells undergoing cellular damage².

Advantages of assay sensitivity

LDH cytotoxicity assays have been proven to deliver high-sensitivity results with a rapid and reliable assessment of cell viability and cytotoxicity. They are simple to perform and can be adapted for testing in various cell types, including 2D and complex 3D culture models such as tumor spheroids^3,7.

Required materials and reagents for LDH assay

An LDH assay requires a standardized kit with key reagents, appropriate lab equipment, and careful storage of assay buffers in aliquots at –20°C to ensure stability and reliable results.

LDH assay kit components

A standardized LDH assay kit includes the following:

• LDH assay buffer and substrate mix containing reagents such as tetrazolium salt/resazurin/ luciferin, 1-methoxyphenazine methosulfate (MPMS), NAD, and lactic acid required for the coupling reaction
• NADH standard control
• LDH positive control
• Stop solution, acetic acid
• Tris base (2-Amino-2-(hydroxymethyl)-1,3-propanediol) and hydrochloric acid (HCl) solution to prepare buffer solutions used as diluent
• Lysis solution, Triton X-100^3,6,12

Essential laboratory equipment

Laboratory equipment required for LDH assay includes

• 96-well clear microplates for sample preparation
• A centrifuge and microcentrifuge tubes for sampling
• Reagent reservoirs, single- and multi-channel pipettes
• A magnetic stir plate for mixing reagents
• A centrifuge with plate adaptors
• An orbital shaker for the missing reaction mixture
• An incubator equilibrated with 5% CO₂ and maintained at 37°C
• Microplate readers such as a spectrophotometer, fluorometer, or luminometer to record the absorbance reading^3,6,12

Storage and handling recommendations

Repeated freezing and thawing of the LDH assay buffer should be avoided. Instead, the assay buffer should be stored in small aliquots (eg, 5 ml for one 96-well microtiter plate) at -20°C for up to 12 months to maintain stability in a dark room^3,6,12.

Experimental protocol for LDH release assay (Colorimetric)

The colorimetric LDH assay involves seeding cells, applying treatments with proper controls, collecting supernatants, adding assay reagents, incubating, and measuring absorbance at 490 nm to quantify cytotoxicity.

Cell culture preparation

• Cells are seeded in a sterile 96-well flat-bottom microtiter plate at a density of 1 × 10⁴ – 5 × 10⁴ cells/well in 100 μL of the culture medium. Samples in each experimental group should be prepared in triplicate wells.
• The cell culture is incubated in a humidified 37°C incubator equilibrated with 5% CO₂ overnight (24 hours).
• Drug treatment is applied as 50-100 μl/well to ensure even mixing so that the sample plates should have a final volume of at least 150 μl/well^6,12.

On the sample plate, the following essential controls should be prepared in triplicates:

• Untreated cells (spontaneous LDH release control) for spontaneous release of LDH from cells.
• Cells treated with lysis buffer (maximum LDH release control) yield an estimate of the maximum LDH activity that could be expected if all cells in the well were killed under the assay conditions.
• Culture medium background (only medium without cells) for LDH activity contributed by serum in the culture medium².

Supernatant collection and reaction setup

• After the incubation period, the microtiter plate should be centrifuged at 1,500-2,000 rpm for 5 min, and 50 μL of the culture supernatant should be collected from each well without disturbing the cell monolayer and transferred to a transparent, untreated assay plate.
• Prepare the buffer solutions and other reagents as per the kit protocol:

o   Buffer A (4mM INT in 0.2 M Tris-HCl, pH 8.2)

o   Buffer B (6.4 mM NAD, 320 mM lithium lactate, in 0.2 M tris-HCl buffer)

o   MPMS supplement (150 mM MPMS in 0.2 M Tris-HCl, pH 8.2)

o   Stop solution (1 M Acetic acid in water)

o   Lysis solution (9% Triton-X100 in water)

o   Diluent (0.2 M Tris-HCl, pH 8.2)

• Prepare the assay reagent by combining equal volumes of buffer A (2.5 ml), buffer B (2.5 ml), and 0.5 μl MPMS supplement per plate. Mix thoroughly to a homogenous light pink or crimson color. Proceed quickly to the next step.
• Add about 50 µL of the assay reagent to each well of the supernatant and mix it in an orbital shaker (300-500 rpm for 15 seconds)^3,6,12.

Incubation time and temperature

The assay plate should be protected from light and incubated at 22-25°C for 30 minutes. Since the LDH assay is a kinetic assay, it is recommended to optimize the incubation time for the cell type to compensate for differing LDH concentrations within the sample. Studies suggest 1 hour incubation to provide maximum spread between experimental wells^3,6,12.

Measurement of absorbance

Add 50 μl/well stop solution to stop the reaction and stabilize the signal. Mix the plate by shaking it for about 30 seconds. Measure the absorbance via a spectrophotometer at 490 nm (INT assay)/450 nm (WST assay). Air bubbles present in wells should be removed using a needle as they hinder the absorbance readings^3,6,12. A reference wavelength (eg, 630-690 nm) should be measured to correct background absorbance and subtract this from the primary wavelength for each well².

Data analysis and interpretation

LDH assay data are analyzed by calculating cytotoxicity using absorbance values, where higher LDH release indicates greater cell damage and a standard curve can optionally provide absolute quantification.

Calculating cytotoxicity

The percentage of cytotoxicity is calculated using the following formula:

Where:

OD _(sample) is the absorbance of test-treated cells

OD _{(spontaneous)} is the absorbance of untreated (negative control) cells

OD _(maximum) is the absorbance of lysis-treated (positive control) cells

The absorbance values used for calculation should be those after background correction^2,12.

Cell viability determination

The effect of conditions on cell viability is determined by comparing the absorbance values between the test and control groups. The quantified absorbance values will be directly proportional to the amount of LDH released in the culture medium. Lower LDH release indicates higher cell viability, while higher LDH release indicates cell cytotoxicity^1,3,7.

Use of standard curve for quantitative analysis (Optional)

To obtain absolute LDH quantification of a sample or estimate cell number or biomass, LDH assays often include calibration of known LDH concentration to create a standard curve.  This allows for the comparison of the unknown sample’s LDH activity to the standard curve, thus determining the concentration of LDH in the sample. This method of quantification is useful in time-course experiments or dose-response studies^2,13.

Applications of LDH assay

Cytotoxicity testing

LDH assays are effective in assessing the effects of environmental factors and drugs on cell viability and measuring the amount of cellular damage¹.

Drug efficacy screening

LDH assays are widely used in cytotoxicity evaluation and cytoprotective studies of drug candidates¹⁴. LDH serves as a potential therapeutic target for malaria and cancer. LDH isoform is a crucial enzyme for ATP generation in the parasite; LDH inhibitors of  Plasmodium falciparum  may be selectively used to target the parasite. The inhibition of LDH-5 has been shown to specifically target the site of tumor progression and invasiveness. An analog of the N-hydroxyindole class of LDH inhibitors has also been effectively tested as an anticancer agent¹.

Monitoring tissue damage

LDH is a ubiquitously expressed enzyme found in nearly all tissues, with particularly high concentrations in the muscle, liver, and kidneys. Its quantification holds significant clinical value, as serum levels of LDH isozymes can indicate tissue-specific pathological conditions. Elevated serum LDH is a hallmark of tissue injury, commonly associated with disorders such as acute myocardial infarction, anemia, pulmonary embolism, hepatitis, and acute renal failure. Pronounced increases in LDH levels are also observed in intracranial hemorrhage, central nervous system lymphoma, leukemia, and metastatic carcinoma¹.

Notably, in metastatic melanoma, in cases of serous effusions, including pleural, pericardial, and peritoneal fluids, elevated LDH levels assist in distinguishing exudates from transudates, thus supporting differential diagnoses. Furthermore, recent research has explored targeting LDHA genes or their protein product, LDH-5, as a potential metabolic intervention strategy in cancer therapy, underscoring LDH’s growing relevance beyond diagnostics¹.

Immunological studies

LDH release assays have proven to be reliable tools for assessing immune cell-induced cytotoxicity, such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, in target cells and the effects of immunotherapy on target cells¹⁵. LDH assays are a valuable tool in evaluating the effectiveness of various immunotherapy-induced cytotoxicity targeting cancer cells and understanding immune-related diseases. Studies have shown that LDH demonstrates an immunosuppressive effect on various immune cells that are becoming ineffective, tolerogenic, and promoting metastasis, angiogenesis, and inflammation¹⁶.

3D cell culture models

LDH assays are successfully utilized for longitudinal monitoring and endpoint analysis of therapeutic efficacy in complex 3D culture models, including spheroids, xenospheres, patient-derived explants (PDE), or organoids^4,14.

Host-pathogen interaction studies

The LDH assay provides valuable insights into studies related to the cytotoxic effects of pathogens (virus, bacteria, parasite) on host cells. Studies have shown that LDH activity in the supernatant of cell culture exposed to infectious agents and/or treatment can be used as a direct measurement of cytotoxicity. It also helps in investigating the infectious disease process and evaluating the efficacy of antimicrobial compounds in host-pathogen interaction studies¹⁷.

Advantages of LDH assays

LDH assays offer a reliable, rapid, and non-radioactive method for detecting cell damage across various cell types, enabling high-throughput, kinetic monitoring of multiple cell death pathways with broad experimental flexibility.

Reliability and stability

LDH is elevated in serum because of organ damage or destruction due to significant cell death that results in loss of cytoplasm. The release of intracellular LDH into the extracellular space or culture medium and its ability to remain stable in the medium make it a valuable tool for assessing cell membrane damage, cell death, and cell viability. The assay is highly sensitive and reliable and can detect low levels of cell damage^3,11.

Ease of use and speed

LDH assays have been proven to provide rapid, robust, and reproducible results in cell viability and cell damage⁴.

High-throughput screening and compatibility

The high-throughput screening and compatibility of LDH assays enable it to further evaluate detailed cytotoxicity determination¹⁸.

Non-radioactive and safer alternative

Measuring the amount of cellular toxicity in a non-invasive manner demonstrates that LDH assays are a safer alternative compared to the Chromium-51 (⁵¹Cr) release assay and are adapted to measure cytotoxic and cytoprotective properties of therapeutic compounds in pharmacology. These assays are harmless for healthy cell populations and can be performed directly in the cell culture wells^14,19.

Broad applicability across cell types

LDH assays are frequently adopted for longitudinal monitoring and endpoint assessment of therapeutic efficacy in 3D culture models such as cell line-derived xenografts and patient-derived explant cultures. This explains their adaptability to different cell types⁴.

Detection of multiple cell death pathways

Studies have shown that the LDH assay can detect low-level damage to the cell membrane as LDH release occurs at the early stage of necrosis. The assay also helps in studying cell death pathways like autophagy, apoptosis, and necrosis. Early LDH measurement is indicative of pyroptotic cell death^11,19.

Assay flexibility and kinetics monitoring

Kinetic LDH assays allow quantitative measurement of absolute LDH activity in the sample supernatant, which favors quantification of cellular injury. It is also versatile and suitable for a wide range of cell types and experimental conditions^11,20.

Comparison with other cell viability assays

Compared to other cell viability assays, LDH assays offer high sensitivity, tissue-specific diagnostic value, non-invasive measurement, and are safer and easier to use with high-throughput compatibility⁷.

LDH assays vs. other assays

Sensitivity and specificity

Due to its ubiquitous presence in all cells, LDH release is highly sensitive and a potential biomarker in the detection of cell damage or cytotoxicity. The LDH isozymes (LDH-1 -LDH-5) each have a specific expression profile in different tissues, which is the basis of their importance as a clinical diagnostic marker. The isozymes vary in molecular structure, substrate affinity, temperature sensitivity, and tissue specificity^1,11.

Troubleshooting and best practices

To ensure accurate LDH assay results, minimize background from serum or hemolysis, use appropriate controls, avoid air bubbles, optimize incubation time per cell type, and properly mix and store reagents.

Common issues

When animal serum is used to supplement the culture medium, it might create a background signal due to endogenous LDH activity interfering with accurate LDH measurement¹⁰. Hemolysis of the blood sample may contribute to an artifactual increase in LDH, leading to false-positive high results¹.

Solutions and recommendations

The use of a serum-free medium or complete media controls, reducing serum concentration in media, may reduce background LDH activity. Performing correct pipetting and preventing air bubbles in the microplate wells prevents inaccurate absorbance readings^10,12.

Optimization tips

Ensure that the reagents are mixed properly during the assay. Preparing and storing assay buffers and reagents and experimenting with the incubation time depending on the cell type play an important role in getting better results¹⁰.

Disadvantages of LDH assays

LDH assays, while useful, have limitations such as non-specificity, serum interference, false positives from hemolysis, reduced stability in long-term or 3D cultures, batch variability in serum, and baseline LDH fluctuations due to normal physiological conditions, necessitating careful controls and complementary methods for accurate interpretation.

Non-specificity as a biomarker

An increased LDH level is an indicator of overall cellular damage but is non-specific as it provides no insight as to the site of tissue injury or mechanism of damage. Combining complementary assays with the LDH assay may provide a clear picture of the cytotoxic effects of a compound, including its impact on different cell death pathways¹¹.

Serum interference

Background interference in the measurement of LDH is generated by serum culture media, which may mask low-level cell damage. Hence, using serum-free media is recommended to avoid this interference^1,10.

False positives due to hemolysis

Improper handling or contamination may cause hemolysis of RBCs in blood samples, resulting in an increased LDH release, which might produce artifactual LDH readings^1,10.

Challenges in long-term or 3D cultures

LDH activity and stability diminish over time, limiting longitudinal monitoring and impeding comprehensive analyses of cellular dynamics over extended timeframes. Additionally, complex 3D culture systems like organoids commonly exhibit high heterogeneity within a single batch, including variations of dimensions, cell densities, cellular contents, and structures, which makes LDH normalization and interpretation difficult¹⁴.

Variability in serum batches

Different serum samples or batches, such as serum containing drugs, acute and chronic conditions, strenuous exercise, age, etc., display variability in basal LDH values. Hence, it is essential to take appropriate care while handling serum cell culture to ascertain reproducible results¹.

Baseline LDH from normal cell turnover

Under normal physiological conditions, such as intensive exercise-induced anaerobic glycolysis, LDH activity is upregulated. Hence, it is vital to maintain controls (maximum LDH release, cell-free culture medium) to define baseline LDH levels for accurate interpretation¹⁰.

LDH assay techniques

LDH assay techniques have evolved with enhanced sensitivity through fluorometric and luminescent assays, improved commercial kits, adaptations for 3D cultures, and multiplexing with other assays, offering more reliable and efficient cell viability and toxicity assessments.

Enhanced sensitivity through fluorometric and bioluminescent assays

Studies demonstrated that fluorometric (silicon quantum dots) and luminescent assays show greater sensitivity and selectivity than traditional colorimetric assays. Luminescent assays enable low-volume sampling and drug-induced toxicity profiling in a time- and dose-dependent manner^9,21.

Improvements in commercial assay kits

Some LDH assay kits offer purified reagents with a longer shelf life and improved signal-to-background ratios. They provide simplified protocols (ready-to-use solutions) to enhance ease of use¹⁴.

Adaptation for 3D cell culture models

It is imperative to adapt and optimize the LDH assay protocol to effectively ensure accurate and reliable viability assessments in 3D cell cultures. Studies have demonstrated normalization of LDH values using bovine serum albumin for stabilizing LDH in the culture medium. Also, optimization of an LDH-preservation buffer, mixed with a conditioned medium at a 1:1 ratio, extends LDH stability for up to a month at −20°C¹⁴.

Multiplexing with other cell-based assays

Another approach is to combine LDH assays with other assays, such as MTT, ATP, Caspase assay, etc, in a single well. Multiplexing has several advantages, such as acquiring data on different parameters simultaneously, including cell viability, cell death mechanisms, cell metabolic activity, and distinguishing between apoptosis and necrosis. It also increases data output, saves time, and is cost-effective^11,22.

Comparison of detection methods

FAQs

What is an LDH assay, and how does it work?

An LDH assay is a cell-based technique that measures LDH enzyme release from damaged or lysed cells. The principle of the LDH assay relies on detecting extracellular LDH activity, which serves as an indicator of cell membrane integrity and cytotoxicity.

How is the LDH cytotoxicity assay performed?

An LDH cytotoxicity assay involves treating cultured cells with a test compound, collecting the supernatant or serum, and measuring LDH activity via a colorimetric or fluorometric reaction; the LDH assay protocol typically includes controls for spontaneous and maximum LDH release to calculate percent cytotoxicity.

What are LDH release assays used for in research?

LDH release assays are widely used in toxicology, drug screening, and immunological studies to evaluate cell damage, cell viability, or immune cell-mediated killing, making them a reliable tool for assessing lactate dehydrogenase release from dying cells.

Which LDH assay kit should I use for cytotoxicity testing?

When selecting an LDH assay kit or LDH cytotoxicity assay kit, consider sensitivity (eg, colorimetric vs. luminescent), compatibility with your cell type, and ease of use; many commercial lactate dehydrogenase assay kits now offer high-throughput formats and improved stability for accurate LDH activity assay results.

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