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Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Lactate dehydrogenase (LDH or LD) is a stable protein present in the cytoplasm of normal cells and normally cannot pass through the cell membrane. When cells are damaged, membrane permeability increases, and LDH is released extracellularly. A decrease in intracellular LDH and an increase in LDH in the culture medium occur. Measuring the LDH activity in the culture medium or the LDH leakage rate can reflect drug-induced cytotoxicity. LDH belongs to the oxidoreductase family and can reversibly catalyze the redox reaction between lactate (L) and pyruvate (P). The reaction formula is: Lactate + NAD⁺ → Pyruvate + NADH + H⁺, where L → P is the forward reaction and P → L is the reverse reaction.
Detection Principle: Using NAD⁺ as a hydrogen acceptor, LDH catalyzes the dehydrogenation of lactate to generate pyruvate. Pyruvate then reacts with dinitrophenylhydrazine to form pyruvate dinitrophenylhydrazone, which appears brownish-red in an alkaline solution. The color intensity is proportional to the pyruvate concentration. The absorbance at 440 nm can be measured using a microplate reader. The released LDH activity during cytotoxicity or the LDH activity in other samples can be calculated using formulas. This kit can be used for routine LDH activity detection and is more commonly used for cytotoxicity assays using LDH release as an indicator.
This kit is for scientific research use only and is not intended for clinical diagnosis or other purposes.
| L1501786 | Component | 100T | 500T | Storage |
| L1501786A | LDH Assay Buffer | 3 mL | 15 mL | 2-8℃. Store in the dark. |
| L1501786B | NAD | 1EA | 2EA | -20℃ |
| L1501786C | Phenylhydrazine Color Solution | 3 mL | 15 mL | 2-8℃. Store in the dark. |
| L1501786D | Alkaline Color Solution | 10 mL | 50 mL | RT. |
| L1501786E | LDH Releasing Agent (10X) | 2 mL | 10 mL | RT. |
User-Prepared Instruments and Reagents
1. 96-well plate cultured test and control group cell samples, sterile PBS, culture medium, distilled water.
2. Microplate centrifuge, 96-well plate or centrifuge, centrifuge tubes, incubator or water bath, microplate reader.
Experimental Procedure
1. Sample Preparation
1.1 LDH Release Assay
Seed an appropriate number of cells into a 96-well culture plate based on cell size and growth rate, so that the cell density does not exceed 90% confluency at the time of detection.
Aspirate the culture medium, wash once with PBS, add fresh culture medium.
Set up corresponding control groups according to experimental needs:
Background Blank Control Well A: Culture medium without cells.
Sample Control Well B: Control cells without drug treatment.
Maximum Enzyme Activity Control Well C: Lysed samples from untreated cells.
Drug-treated Sample Well D: Cells treated with the drug.
Continue cultivation.
Before detection, take out the cell culture plate. Add LDH Releasing Agent (10X) to the "Maximum Enzyme Activity Control Well C" at a volume equal to 10% of the original culture medium volume. Mix thoroughly by pipetting up and down several times. Continue cultivation for about 1 hour.
Centrifuge the cell culture plate at 400 g for 5 minutes using a microplate centrifuge.
Aspirate 5 µL of supernatant from each well and transfer it to the corresponding wells of a new 96-well plate for subsequent LDH detection.
1.2 Cytotoxicity and Cell Proliferation Assay for Intracellular Total LDH
Seed an appropriate number of cells into a 96-well culture plate based on cell size and growth rate, so that the cell density does not exceed 90% confluency at the time of detection.
Treat with different drugs and set up appropriate controls.
Centrifuge the cell culture plate at 400 g for 5 minutes using a microplate centrifuge.
Aspirate the culture medium.
Add 150 µL of LDH Releasing Agent diluted 10-fold with PBS. Shake the plate to mix thoroughly. Continue cultivation for about 1 hour.
Centrifuge the cell culture plate at 400 g for 5 minutes using a microplate centrifuge.
Aspirate 5 µL of supernatant from each well and transfer it to the corresponding wells of a new 96-well plate for subsequent cytotoxicity detection.
1.3 Protein Concentration Determination
After sample preparation, the protein concentration can be determined using a BCA Protein Assay Kit (Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit are recommended) to facilitate subsequent calculation of LDH content per unit protein weight in tissues or cells.
2. Preparation of NAD Solution
Take one vial of NAD (powder) and dissolve it in 1.5 mL of deionized water.
3. LDH Enzymatic Reaction
Add solutions sequentially according to the table below, taking care to avoid bubbles. If the enzyme activity in the sample is too high, reduce the sample volume or dilute appropriately before assay.
| Reagent (µL) | Volume (µL) |
| Test Sample (supernatant) | 5 |
| LDH Assay Buffer | 25 |
| NAD Solution | 5 |
Mix well, incubate at 37°C for 15 min.
| Phenylhydrazine Color Solution | 25 |
Mix well, incubate at 37°C for 15 min.
| Alkaline Color Solution | 100 |
| Distilled Water | 150 |
4. LDH Measurement
Mix well and let stand at room temperature for 5 minutes. Measure the absorbance of each well at 440 nm using a microplate reader.
5. Result Calculation
Cytotoxicity or Mortality Rate (%) = (A D - A B ) / (A C - A B ) × 100%
If the absorbance value A γ of a known concentration *c* of an LDH enzyme standard and the absorbance value A γ0 of the standard blank control are measured simultaneously, the enzyme activity in the sample can be roughly calculated:
LDH Activity in Test Sample (mU/mL) = (A B - A A ) / (A γ - A γ0 ) × *c*
For accurate calculation of the absolute LDH enzyme activity in the sample, use a self-prepared LDH standard to plot a standard curve with the measured absorbance values. The enzyme activity of the sample can be calculated using the formula derived from the standard curve.
Where:
A A = Absorbance of Background Blank Control Well A
A B = Absorbance of Sample Control Well B
A C = Absorbance of Maximum Enzyme Activity Control Well C
A D = Absorbance of Drug-treated Sample Well D
6. Results and Analysis
The cytotoxicity of drugs or toxicants can be determined by directly comparing the LDH activity in each well. Higher LDH activity indicates higher cell membrane permeability and more severe cell damage.
Precautions
1. Use serum-free or low-serum concentration culture medium when culturing cells to exclude serum interference; otherwise, deviations may occur.
2. EDTA inhibits LDH. Avoid using or thoroughly remove reagents containing EDTA during operation.
3. Measure LDH as soon as possible after collection. If the collected cell culture medium is stored for too long, LDH activity may decrease.
4. Use solutions prepared at the same time for the same batch of experiments. The volume of solutions used and the reaction time should be consistent.
5. In the enzymatic reaction, the recommended supernatant sample volume is 2.5-10 µL. If the enzyme activity in the sample is too high, reduce the sample volume or dilute appropriately before assay.
6. Measurement should be completed within 15 minutes after color development.
7. The Alkaline Color Solution is somewhat corrosive; handle with care.
8. Use reagents promptly after opening to avoid affecting subsequent experimental results.
9. For your safety and health, please wear a lab coat and disposable gloves during operation.
Comprehensive hazard, handling, storage, and regulatory compliance document.
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