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| Product Name | Acid Phosphatase Cell Cytotoxicity Colorimetric Assay Kit |
| Catalog No. | CATR-HMM-0055 |
| Description | A colorimetric cytotoxicity assay based on the measurement of acid phosphatase (ACP) activity in viable adherent cells. The assay uses p-nitrophenyl phosphate (pNPP) as a chromogenic substrate that is hydrolyzed by intracellular acid phosphatases in viable cells to produce yellow p-nitrophenol, which is measured at 405 nm. This method provides a simple, low-cost alternative to MTT/XTT assays for adherent cell viability and cytotoxicity assessment. |
| Intended Use | Cytotoxicity screening with adherent cell lines; anti-cancer drug efficacy testing; biomaterial cytotoxicity evaluation (ISO 10993-5); high-throughput adherent cell viability quantification; low-cost viability assessment in resource-limited settings. |
| Principle / Technology | Intracellular acid phosphatases in viable, adherent cells hydrolyze p-nitrophenyl phosphate (pNPP) to p-nitrophenol (yellow) in acidic buffer; color development is proportional to viable adherent cell number |
| Detection Method | Colorimetric; absorbance at 405–410 nm; reference wavelength 620 nm |
| Sample Type | Adherent mammalian cells; not suitable for suspension cells or weakly adherent cell types |
| Sensitivity / LOD | Linear range 500–50,000 cells per well in 96-well format; LOD approximately 200 cells |
| Specificity | Specific to viable adherent cells; dead cells detach and are removed during wash steps; sensitivity comparable to MTT assay |
| Reaction Conditions / Protocol | Remove medium; wash cells with PBS; add 100 µL assay buffer with pNPP substrate; incubate 1–2 hours at 37 °C; add 10 µL 1 N NaOH stop solution per well; read absorbance at 405 nm |
| Components / Formulation | pNPP substrate tablets (10× 20 mg), citrate buffer pH 5.5 with 0.1% Triton X-100 (assay buffer), stop solution (1 N NaOH), 96-well plate sealing film |
| Storage Conditions | Store at -20 °C; pNPP tablets stable at -20 °C protected from moisture; assay buffer at 4 °C |
| Shelf Life | 12 months from manufacture date |
| Package Specifications | 500 tests, 1,000 tests, 5,000 tests in 96-well format |
| Product Form | Tablet substrate with liquid buffers |
| Key Features | Low-cost cytotoxity assay — approximately 10× less expensive than MTT per test; simple add-and-read protocol with stable endpoint; no solubilization step required (unlike MTT); non-toxic reagents (unlike MTT/formazan); compatible with standard absorbance plate readers; validated per ISO 10993-5 for biomaterial testing |
| Purity | pNPP substrate purity ≥99% by HPLC; phosphatase substrate certified |
| Concentration | As specified on product label; working concentrations optimized per protocol |
| Activity / Unit Definition | Signal-to-noise ratio and linearity verified on reference cell lines per lot |
| Molecular Weight | Varies by dye or reagent component as specified |
| Source / Origin | Synthetic dyes and tetrazolium compounds; recombinant enzymes where applicable |
| pH Range / Optimal pH | pH 7.2–7.4 for cell-based assays |
| Shipping Conditions | Cold pack -20 °C for substrate tablets; assay buffer at ambient or 4 °C |
| Expiration Date / Stability | 12 months at -20 °C; reconstituted pNPP substrate solution stable for 2 weeks at 4 °C or 2 months at -20 °C; protect all reagents from light |
| Regulatory / Compliance | For laboratory and research use only; RUO; manufactured under ISO 9001; pNPP is not classified as dangerous goods |
| Compatibility | Compatible with adherent cells cultured in standard 96-well and 24-well TC-treated plates; assay buffer contains Triton X-100 for cell permeabilization; sodium azide and other phosphatase inhibitors in culture medium must be washed out before assay |
| Recommended Buffer System | PBS or phenol red-free complete medium as specified in protocol |
| Application Notes / Precautions | Wash cells gently with PBS to avoid detaching viable cells; two gentle PBS washes recommended. Include cell-free blank wells and untreated control wells. For drug treatment studies, compounds that inhibit phosphatases may produce false-positive cytotoxicity results — confirm with alternative assay. Incubation time may need optimization: rapidly proliferating cells (30–60 min), primary cells (1–2 hours). |
| Batch-to-Batch Consistency | Signal linearity R² ≥0.99 with reference cell number standard curve per lot |
For research use only, not for clinical use.
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