UA-Glo® LDH Luminescence Cytotoxicity Assay

Lactate dehydrogenase (LDH) is widely present in the cytoplasm of cells and is released extracellularly upon cell membrane rupture, remaining active in cell culture media for extended periods. Therefore, extracellularly released LDH is often used as an indicator of cellular health to reflect the toxic effects of external stimuli or compounds on cells. The UA-Glo® LDH Luminescent Cytotoxicity Assay Kit measures the extracellular LDH content through a coupled reaction involving LDH, reductase, and luciferase to assess cellular health.

The UA-Glo® LDH Luminescent Cytotoxicity Assay includes reductase, luciferase, and enzyme substrates, where the reductase substrate also serves as a precursor for luciferase substrate luciferin. The LDH reaction converts NAD to NADH, and the reductase utilizes NADH to convert the reductase substrate into luciferin, which is then used by luciferase to generate luminescence. The luminescence intensity is directly proportional to LDH activity, thereby indirectly determining cellular health. The UA-Glo® LDH Luminescent Cytotoxicity Assay features high signal-to-noise ratio, excellent reproducibility, and stability. Its homogeneous, ready-to-use formulation requires only a single addition, reducing potential experimental errors from multiple pipetting steps. Additionally, this assay does not require cell lysis, enabling continuous monitoring of the same cell sample and further simplifying experimental design.

The UA-Glo® LDH Luminescent Cytotoxicity Assay can be combined with other cellular health assessment reagents for multiplex detection on the same experimental sample, such as the UA-Glo® CTG Cell Viability Assay Kit (Cat# UA070103), Caspase 3/7 Apoptosis Assay Kit (Cat# UA079012), and CTF Fluorescent Cell Viability Assay Kit (Cat# UA079015), to better elucidate the mechanisms of cytotoxicity.

UA-Glo® LDH Luminescent Cytotoxicity Assay Kit components and specifications are as follows. The detectable number of reactions for 96-/384-well plates is calculated based on adding 50/10 μL of sample and 50/10 μL of LDH detection reagent per well, respectively.

# Translation Result

UA-Glo® LDH luminescence cytotoxicity assay kit measures extracellular LDH levels through a coupled reaction involving LDH, reductase, and luciferase to assess cell health. The luciferase reaction offers extremely high sensitivity, but parameters such as pH, temperature, and experimental setup significantly affect the results. Direct comparison of absolute readings is not recommended under most experimental conditions. It is advisable to include the same reference controls on each plate—for example, a cytotoxic reference compound and solvent vehicle control in tumor cell cytotoxicity assays—and normalize sample readings against these controls before data processing and comparison. The cytotoxicity (%) in the UA-Glo® LDH luminescence assay can be calculated using the following formula: Cytotoxicity (%) = 100 × (Sample reading – Solvent vehicle control reading) / (Maximum LDH release control reading – Solvent vehicle control reading). The maximum LDH release control can be either a cytotoxic reference compound or a solvent vehicle control treated with Triton X-100 at a final concentration of 0.2% to lyse cells for 10–15 min before supernatant collection.

The kit includes a tube of LDH positive control for reagent validation and determination of the linear detection range. The LDH positive control can be diluted from the LDH stock solution (see Section 1.4) to 30 mU/mL, which is the highest concentration. A 2× serial dilution can then be performed from 30 mU/mL in 7–8 steps to generate a standard curve.

The UA-Glo® LDH luminescence cytotoxicity assay kit is suitable for cytotoxicity testing in tumor cells, primary cells, 3D cell models, stem cells, and ADCC assays. Below is an example protocol for a compound cytotoxicity assay in a 96-well plate.

1.Sample Preparation:

1)Seed experimental cells at an appropriate density in a 96-well plate with 100 µL medium per well.

2)Treat cells with compounds as required. Include a cytotoxic reference compound and solvent vehicle control on each plate for normalization.

3)Collect samples for LDH measurement at multiple time points post-treatment, if needed.

4)For sampling, take 2–5 µL of cell culture medium and dilute 20× withLDH stock solution (200 mM Tris-HCl, pH 7.3, 10% glycerol, 1% BSA). Diluted samples can be tested immediately or stored at –20°C or below until analysis.Do not dilute LDH with medium or buffers (e.g., PBS), as this will significantly reduce LDH activity.

2.LDH Luminescence Cytotoxicity Assay

1)Prepare the LDH detection reagent freshly before use (steps 2)–4)). For a 96-well plate, prepare enough reagent for 50 µL per sample (total 100 µL reaction volume). For a 384-well plate, prepare 10 µL per sample.

2)Thaw the UA-Glo® LDH detection solution and reductase substrate at room temperature (22–25°C), then place the reductase substrate on ice.

3)After complete thawing, equilibrate the LDH detection solution to room temperature and mix thoroughly. Luciferase reactions are sensitive to temperature fluctuations; ensure reagents and samples are equilibrated, and maintain a constant temperature (±1°C) during testing.

4)The reductase substrate is 200× concentrated; dilute to 1× in LDH detection solution to prepare theLDH detection reagent. Mix thoroughly. After first use, aliquot the reductase substrate and store protected from light at –80°C to avoid repeated freeze-thaw cycles.

5)Retrieve the 20× diluted samples. For most experiments, further dilute samples 2×–5× with LDH stock solution (final dilution: 40×–100×) before testing. Optimal dilution may vary depending on experimental parameters (e.g., cell number, serum content in medium—some serum brands contain high LDH). Pre-testing is recommended to ensure readings fall within the linear range. Refer to the "Sample Dilution Guide" table below for guidance.

6)Add 50 µL of diluted sample per well in a white (preferably opaque-bottom) 96-well plate, followed by 50 µL of LDH detection reagent.

7)Mix by shaking at medium speed for 1 min, then incubate in the dark for 30–60 min.

8)Measure luminescence using a plate reader. Readings can be taken after 60 min, but ensure signals remain within the linear range and are still increasing.

Sample Dilution Guide

1) Different batches are not recommended for mixed use 2) Without strict validation, it is not recommended to alter the dosage of detection reagents 3) For research use only