LDH assay: Principles, protocols, and applications

Explore LDH assay kits for cytotoxicity and cell viability analysis. Learn principles, protocols, and applications with Abcam. Lactate dehydrogenase (LDH) assays quantify the release of LDH from damaged cells into the cell culture supernatant.

LDH is a commonly used method for determining cell viability and cytotoxicity following cell damage or death, including necrosis, apoptosis, and other forms of tissue damage. LDH release assays are widely applied in research, particularly during pre-clinical drug development and in assessing cellular responses to therapeutic treatments^1,2.

The LDH release assay offers a fast and reliable approach for quantifying elevated LDH levels, serving as a surrogate marker of cellular damage. It provides rapid, reproducible results and is widely used to evaluate cytotoxic effects of therapeutic compounds across diverse biological samples³.

LDH activity can be detected in plasma, serum, tissues, cells, and culture media. Commercial LDH assay kits are adapted for longitudinal monitoring of cell growth and viability in 2D and 3D systems, including xenospheres, patient-derived explants (PDEs), and spheroids⁴.

Biochemical principles of LDH assay

Under normal physiological conditions, glucose metabolism proceeds via glycolysis, generating pyruvate that is further oxidized through oxidative phosphorylation to produce ATP. In anaerobic or hypoxic conditions, LDH activity increases to sustain energy production through anaerobic glycolysis^1,5.

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 transfers a hydride ion from NADH to pyruvate, ensuring NAD⁺ regeneration required for continuous glycolytic flux.

The LDH cytotoxicity assay uses this enzymatic activity to measure cell membrane integrity. When cells are damaged or lysed, LDH is released into the extracellular environment, where its activity can be detected and quantified using coupled enzymatic reactions.

In this assay, LDH converts lactate to pyruvate while reducing NAD⁺ to NADH. NADH drives the reduction of chromogenic or fluorogenic substrates such as tetrazolium salts, resazurin, or luciferase systems, producing measurable signals proportional to LDH release.

The signal intensity detected in the culture supernatant directly correlates with LDH release, serving as an indicator of cell viability or cytotoxicity. This tissue-wide applicability is supported by the ubiquitous expression and multiple isozyme forms of LDH^1,3,6.

Types of LDH assays

LDH assays include colorimetric, fluorometric, and bioluminescent formats. These methods differ in sensitivity, detection mechanisms, and suitability for evaluating cell viability and cytotoxicity, enabling researchers to select appropriate approaches based on experimental requirements.

Colorimetric LDH assays

Colorimetric assays quantify cytosolic LDH release using dye-based biochemical reactions to assess metabolic activity. LDH activity is measured by absorbance using a spectrophotometer, making these assays widely used for their simplicity, safety, and cost-effectiveness.

In these assays, LDH catalyzes the oxidation of lactate to pyruvate, generating NADH, which reduces tetrazolium salts to formazan. The resulting water-soluble dye can be quantified spectrophotometrically, reflecting the degree of cellular damage in the culture medium^2,7,8.

For example, iodonitrotetrazolium chloride (INT) is reduced to a red formazan dye, which is measurable at 492 nm. The absorbance is directly proportional to cell damage. Various tetrazolium salts are used across assays for different sensitivity and performance characteristics.

Colorimetric assays also include MTT, MTS, XTT, WST-1, WST-8, sulforhodamine B (SRB), neutral red uptake (NRU), and crystal violet assays⁷. These methods collectively provide versatile tools for examining cell viability and proliferation.

Fluorometric LDH assays

Fluorometric assays offer enhanced sensitivity, broader linear range, and higher throughput compared to colorimetric methods. These properties make them suitable for detecting low levels of LDH activity and for applications requiring greater assay precision.

A common example is the resazurin reduction assay, in which resazurin is converted to the fluorescent resorufin. LDH generates NADH, which facilitates this conversion, producing fluorescence that correlates with metabolic activity and cellular viability.

The fluorescence intensity of resorufin, typically measured at 560–590 nm, is proportional to cellular metabolic activity. This method enables sensitive detection and is frequently used in high-throughput screening applications^7,9.

Bioluminescent LDH assays

Bioluminescent assays are the most sensitive and rapid methods for detecting LDH activity. These assays generate luminescent signals via luciferase reactions, enabling highly sensitive measurements even from small sample volumes.

In this assay, NADH production drives conversion of pro-luciferin to luciferin, producing light measurable using a luminometer. The luminescent signal remains stable after reagent addition, enabling reliable detection and analysis.

These assays enable detection with small sample sizes and measurement of subtle changes in cell viability. They are widely used in complex 3D culture systems and advanced biological models^7,10,11.

Mechanism of the LDH assay

LDH release and detection

Exposure to cytotoxic conditions disrupts cell membrane integrity, leading to necrosis, apoptosis, or other forms of cell death. Cytoplasmic contents, including LDH, leak into the extracellular environment, where they can be detected for analysis.

Due to its stability and persistence in circulation for up to 7 days, as well as its presence in all cell types, LDH serves as a reliable biomarker for assessing cell damage and cytotoxicity¹.

Coupled enzyme reactions

Elevated LDH catalyzes the conversion of pyruvate to lactate and NAD⁺ to NADH. NADH then reduces substrates such as resazurin or tetrazolium salts, generating measurable signals through coupled enzymatic reactions.

These signals, including colorimetric or luminescent outputs, are proportional to LDH activity. Quantification reflects the extent of cellular damage and the number of affected cells, enabling precise cytotoxicity determination.².

Advantages of assay sensitivity

LDH cytotoxicity assays provide high sensitivity and rapid, reliable measurement of cell viability. They are simple to perform and adaptable to different cell types, including both 2D monolayers and complex 3D culture models such as tumor spheroids^3,7.

Figure 1. Diagram showing the principles of the Lactate Dehydrogenase assay method. LDH Assay Kit / Lactate Dehydrogenase Assay Kit (Colorimetric)

Required materials and reagents for the LDH assay

An LDH assay requires standardized kits, appropriate laboratory equipment, and proper buffer storage conditions. Maintaining reagents in aliquots at −20°C ensures stability and consistent assay performance over extended periods.

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.

1. 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.

2. 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.

3. 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.

4. 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 to estimate cell number or biomass, LDH assays often include calibration with a known LDH concentration to create a standard curve.

This allows comparison of the unknown sample’s LDH activity with the standard curve, thereby determining the LDH concentration 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 (PDEs), or organoids^4,14.

Host-pathogen interaction studies

The LDH assay provides valuable insights into the cytotoxic effects of pathogens (viruses, bacteria, parasites) on host cells. Studies have shown that LDH activity in the supernatant of cell cultures exposed to infectious agents and/or treatments can be used as a direct measure of cytotoxicity. It also helps investigate the infectious disease process and evaluate the efficacy of antimicrobial compounds in host-pathogen interaction studies 17.

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

LDH cytotoxicity

MTT

WST

ATP

Reduction of INT resulting in red formazan, which is measured quantitatively at 490 nm.

MTT is reduced to purple formazan by NADH.

WST-1 is converted to formazan by mitochondrial dehydrogenase enzymes in the presence of mPMS.

Luciferin is converted to oxyluciferin by luciferase in the presence of Mg²⁺ ions and ATP, yielding a luminescent signal.

Applied for assaying cell-mediated cytotoxicity and cytotoxicity mediated by toxic chemicals and drugs.

Determines cell viability by measuring the activity of mitochondrial enzymes, eg, succinate dehydrogenase.

Used to measure the relative rate of cell proliferation in the culture.

Measures ATP concentration and cell number by reading the intensity of the luminescent signal.

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

Colorimetric

Fluorometric

Luminometric

Simple and easy to perform, cost-effective, sensitive

More sensitive than a colorimetric assay

Most sensitive method and enables sampling of 2-5 µl of the culture supernatant at various time periods

Assessment of cell viability and cytotoxicity

High throughput and wide linearity range

Samples can be stored frozen for future analysis of the trends of LDH release over time

Versatile and suitable for a wide variety of cell types and experimental conditions

Offers good biocompatibility, low toxicity, good water solubility, and photostability

Rapid and easy to perform, providing results within a short time frame, and an excellent choice for high-throughput screening in large-scale studies

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.