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Cytotoxicity LDH Assay Kit

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MCE Cytotoxicity LDH Assay Kit can detect LDH activity in the culture supernatant, which is indicative of cytotoxicity.

  • Storage :

    Stored at -20°C protecting from light, and is stable for up to 12 months.

  • Experimental Validation Results for Cytotoxicity LDH Assay Kit
  • Experimental Validation Results for Cytotoxicity LDH Assay Kit
  • Experimental Validation Results for Cytotoxicity LDH Assay Kit
  • Experimental Validation Results for Cytotoxicity LDH Assay Kit
  • Experimental Validation Results for Cytotoxicity LDH Assay Kit
  • Cytotoxicity LDH Assay Kit Product Image
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Description & Advantages

The loss of cell membrane integrity due to cell death or membrane damage results in release of certain soluble, cytosolic enzymes. Therefore, acute cytotoxicity can be measured by measuring activity of these enzymes released into the culture supernatant. Lactate dehydragenase (LDH) is a stable enzyme, present in all cell types, and rapidly released into the cell medium upon damage of the plasma membrane. LDH, therefore, is the most widely used marker in cytotoxicity study.

 

LDH oxidizes lactate to generate NADH, which then reacts with certain dye to generate yellow color. The intensity of the generated color correlates directly with the cell number lysed, which is indicative of cytotoxicity. LDH activity can be easily quantified by spectrophotometer or plate reader at 490 nm.

Fig 1. Schematic of MCE Cytotoxicity LDH Assay Kit.

MCE Cytotoxicity LDH Assay Kit can be directly added to cells to detect LDH activity in the culture supernatant (one-step, direct method). Or you can separate the culture supernatant from cell and use this kit to detect LDH activity (indirect method). The separated cells can be used for other experiments.

Fig 2. Detect LDH activity by direct and indirect method.

Protocol

1. 96-well plate setup

Well group Treatment
Medium background Medium only, no cells
Low control (spontaneous release) Cells + medium/vehicle
High control (maximum release) Cells + Lysis Solution
Test sample Cells + test treatment

1. Culture and treat cells according to the experimental design. Add an appropriate amount of Lysis Solution to the high-control wells before the assay to achieve maximum LDH release.

2. After treatment, briefly centrifuge the plate to sediment cells and debris.

2. LDH colorimetric reaction

1. Transfer 50 μL culture supernatant from each well to a new 96-well plate.

2. Add 50 μL Working Solution to each well and mix gently.

3. Incubate for 30 min at room temperature protected from light.

4. Add 50 μL Stop Solution to each well and mix gently.

5. Measure absorbance at 490 nm. A reference wavelength of 600-690 nm may be used for background correction.

3. Calculation of cytotoxicity

Cytotoxicity (%) = (A490 of test sample - A490 of low control) / (A490 of high control - A490 of low control) × 100%

Note: Subtract medium background from all wells and ensure sample readings fall within the valid range defined by the high and low controls.

Storage

Stored at -20°C protecting from light, and is stable for up to 12 months.

Components

Components HY-K1090-100T HY-K1090-500T
Working Solution 5.5 mL 27.5 mL
Stop Solution 5.5 mL 27.5 mL
Lysis Solution 1.1 mL 5.5 mL

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  • Experimental Validation Results for Cytotoxicity LDH Assay Kit
  • Experimental Validation Results for Cytotoxicity LDH Assay Kit
  • Experimental Validation Results for Cytotoxicity LDH Assay Kit
  • Experimental Validation Results for Cytotoxicity LDH Assay Kit
  • Experimental Validation Results for Cytotoxicity LDH Assay Kit
  • Cytotoxicity LDH Assay Kit Product Image