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| Product Name | Cytotoxicity Detection Kit PLUS, LDH Release, Water-Soluble Tetrazolium Salt |
| Catalog No. | CATR-HMM-0062 |
| Description | Colorimetric cytotoxicity assay kit based on lactate dehydrogenase (LDH) release from damaged cells into culture supernatant. The coupled two-step enzymatic reaction converts LDH-catalyzed lactate oxidation (with NAD+ reduction to NADH) and subsequent reduction of the water-soluble tetrazolium salt WST-1 to a colored formazan dye (absorbance 440-490 nm). The formazan product is water-soluble, eliminating the dissolution step required by MTT and XTT assays. The amount of formazan dye formed is proportional to LDH activity released from dead cells, enabling quantitative cytotoxicity measurement without pre-labeling or washing steps. |
| Intended Use | Quantitative measurement of cytotoxicity, cell death, and membrane integrity loss in response to drug treatments, toxins, immune effector cells, and environmental stress in cell-based assays. |
| Principle / Technology | LDH released from damaged cells catalyzes lactate → pyruvate conversion with concomitant NAD+ → NADH reduction; NADH reduces WST-1 (water-soluble tetrazolium) to colored formazan via electron mediator; absorbance proportional to dead cell number. |
| Detection Method | Absorbance microplate reader at 440-490 nm (reference wavelength >600 nm); endpoint measurement after 15-30 minute color development. |
| Sample Type | Cell culture supernatants from adherent and suspension cells; co-culture media; serum-containing or serum-free media. |
| Performance Range / Specifications | Linear range: 0.1-10% cytotoxicity (HeLa cells, 10,000 cells/well); equivalent to detection of 10-1,000 dead cells per well in 96-well format; total LDH release by Triton X-100 lysis serves as 100% reference. |
| Sensitivity / LOD | Detection of LDH from as few as 100 dead HeLa cells per well; formazan absorbance 0.02-2.5 AU at 450 nm. |
| Specificity | LDH-catalyzed reaction is specific for lactate substrate; WST-1 reduction is specific for NADH via electron mediator; phenol red in culture medium does not interfere at recommended measurement wavelength. |
| Reaction Conditions / Protocol | Transfer 100 µL culture supernatant to assay plate; add 100 µL LDH reaction mixture; incubate 15-30 min at RT in dark; read absorbance at 450 nm. |
| Components / Formulation | WST-1/diaphorase/NAD+ mixture (lyophilized), LDH positive control (porcine heart), Triton X-100 lysis solution (10% v/v), lactate substrate solution, detailed protocol. |
| Storage Conditions | Store WST-1 mixture at -20 °C protected from light; LDH control at -20 °C; other reagents at 2-8 °C. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 500 tests, 2000 tests, 5000 tests (96-well format). |
| Product Form | Lyophilized powder (WST-1 mix); liquid reagents; reconstitute with assay buffer. |
| Quality Control | Each lot tested for linearity of LDH activity standard curve (0-5 mU/well); formazan formation rate verified; LDH positive control activity certified. |
| Key Features | Water-soluble formazan product (no solubilization step); homogeneous add-mix-read protocol; non-radioactive; compatible with serum-containing media; phenol red tolerant; suitable for high-throughput screening. |
| Purity | WST-1 purity ≥98%; diaphorase recombinant enzyme; LDH control specific activity 200-400 U/mg. |
| Concentration | Reaction mixture: prepare fresh before each use at 1× concentration. |
| Activity / Unit Definition | LDH positive control: 10 U/mL stock; use 1-10 mU per well for standard curve. |
| Molecular Weight | WST-1: 751.71 g/mol (C32H27N7NaO6S2). |
| Source / Origin | Recombinant diaphorase; LDH from porcine heart tissue. |
| pH Range / Optimal pH | Assay buffer pH 8.0-8.5; optimal LDH activity at pH 8.2. |
| Shipping Conditions | Cold pack; WST-1 mixture on dry ice recommended for long-distance transit. |
| Expiration Date / Stability | 12 months at -20 °C; reconstituted WST-1 mixture stable for 2 weeks at 2-8 °C protected from light. |
| Regulatory / Compliance | For research use only; not for clinical diagnostic procedures. |
| Compatibility | Compatible with DMEM, RPMI-1640, MEM, and other standard media including phenol red. Hemoglobin at >50 µg/mL may cause interference due to pseudoperoxidase activity — centrifuge samples if erythrocyte contamination is present. |
| Recommended Buffer System | Lactate substrate in Tris buffer, pH 8.2. |
| Application Notes / Precautions | Include cell-free medium controls (background LDH) and Triton X-100 lysed controls (maximum LDH release) for each plate. Centrifuge supernatant at 250 × g for 5 minutes to remove cell debris before transfer. For kinetic measurements, read absorbance every 5 minutes over 30 minutes and calculate rate. Protect reaction mixture from light during incubation. Complete lysis with 1% Triton X-100 requires 30-45 minutes at 37 °C. |
| Batch-to-Batch Consistency | WST-1 reduction rate within ±10% of reference lot; LDH standard curve slope within ±10%. |
For research use only, not for clinical use.
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