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| Product Name | Senescence-Associated Beta-Galactosidase (SA-β-Gal) Detection Kit |
| Catalog No. | CCAT-HMM-0070 |
| Description | Histochemical staining kit for detection of senescence-associated β-galactosidase (SA-β-Gal) activity in cultured cells and tissue sections at pH 6.0 — a widely validated biomarker of cellular senescence. Senescent cells express elevated lysosomal β-galactosidase activity detectable at suboptimal pH 6.0 due to increased lysosomal mass and enzyme content, whereas normal proliferating or quiescent cells show negligible activity at this pH. The X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) chromogenic substrate produces an insoluble blue precipitate within SA-β-Gal-positive cells, enabling direct visualization and quantification by light microscopy. |
| Intended Use | Histochemical detection and quantification of senescent cells in adherent cell cultures, frozen tissue sections, and tissue biopsies for cellular senescence, aging, tumor suppression, and senescence-associated secretory phenotype (SASP) research. |
| Principle / Technology | β-galactosidase cleaves X-Gal substrate at pH 6.0 releasing 5-bromo-4-chloro-3-hydroxyindole that dimerizes and oxidizes to insoluble blue indigo precipitate; reaction is specific for senescent cells due to elevated lysosomal enzyme content; pH 6.0 optimizes discrimination between senescent and non-senescent cells. |
| Detection Method | Light microscopy (bright-field); blue-stained cells counted as SA-β-Gal positive; percentage of positive cells calculated from total cell count. |
| Sample Type | Adherent cultured cells (primary fibroblasts, epithelial cells, cancer cell lines after senescence induction); frozen tissue sections (5-10 µm). |
| Performance Range / Specifications | Detects 1-100% senescent cells in a population; staining visible within 2-4 hours for strongly positive cells; overnight incubation for weakly senescent populations. |
| Sensitivity / LOD | Detection of senescent cells at >5% prevalence in population (based on 200 cells counted); SA-β-Gal positive cells distinguishable from background by distinct perinuclear blue granular staining. |
| Specificity | SA-β-Gal activity at pH 6.0 distinguishes senescent from quiescent (serum-starved) and proliferating cells; false positives may occur in confluent contact-inhibited cultures and cells with high endogenous lysosomal activity. |
| Reaction Conditions / Protocol | Wash cells with PBS; fix with Fixative Solution 10-15 min at RT; wash with PBS; add Staining Solution (X-Gal + Staining Supplement + Staining Buffer, pH 6.0); incubate at 37 °C without CO2 for 2-24 hours protected from light; examine under microscope; store plates at 2-8 °C for up to 1 week. |
| Components / Formulation | X-Gal substrate (20 mg/mL in DMF), Staining Supplement (20×), Fixative Solution, Staining Buffer (pH 6.0), detailed protocol. |
| Storage Conditions | Store X-Gal at -20 °C; Fixative Solution at room temperature; Staining Supplement and Buffer at 2-8 °C. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 1 kit (sufficient for ~50 staining reactions in 35 mm dishes), 2 kits. |
| Product Form | X-Gal: colorless DMF solution; Staining Supplement: concentrated aqueous solution; Fixative Solution: clear liquid; Staining Buffer: clear liquid. |
| Quality Control | Each lot tested for SA-β-Gal staining in irradiated (10 Gy) senescent WI-38 fibroblasts vs. proliferating WI-38 cells (>90% positive vs. <5% positive); X-Gal substrate activity verified. |
| Key Features | Classic histochemical senescence detection; blue precipitate for direct visualization; includes optimized fixative and staining supplement; validated at pH 6.0; compatible with cell imaging and quantification software; cost-effective. |
| Purity | X-Gal ≥98% by HPLC; DMF anhydrous grade. |
| Concentration | X-Gal working concentration: 1 mg/mL in Staining Solution. |
| Activity / Unit Definition | β-galactosidase activity detected at pH 6.0; lysosomal enzyme activity optimal at pH 4.0-4.5 but shifted to pH 6.0 for senescence specificity. |
| Molecular Weight | X-Gal: 408.63 g/mol (C14H15BrClNO6). |
| Source / Origin | Synthetic X-Gal chromogenic substrate; laboratory-grade buffer salts. |
| pH Range / Optimal pH | Staining Solution pH 6.0 ± 0.1 (critical for senescence specificity). |
| Shipping Conditions | Ambient temperature; X-Gal on cold pack recommended. |
| Expiration Date / Stability | 12 months at recommended storage; X-Gal solution stable for 3 months at -20 °C after opening. |
| Regulatory / Compliance | For research use only; not for diagnostic or clinical applications. |
| Compatibility | Compatible with most adherent mammalian cell types. Not recommended for suspension cells or cells grown on non-adherent surfaces. Fixation is critical — over-fixation (>15 minutes) may reduce enzyme activity. DMF in X-Gal solution is toxic — work in fume hood when preparing staining solution. |
| Recommended Buffer System | Citrate-phosphate buffer, pH 6.0, with potassium ferrocyanide and potassium ferricyanide as oxidation catalysts, magnesium chloride as enzyme cofactor, and sodium chloride for tonicity. |
| Application Notes / Precautions | Verify pH of staining solution is exactly 6.0 — even ±0.2 pH unit shifts can reduce discrimination between senescent and non-senescent cells. Include positive control (irradiated or replicative senescent cells) and negative control (early passage proliferating cells at <50% confluence). Do not let cells become over-confluent before staining — contact inhibition can induce false-positive SA-β-Gal. For co-staining, perform SA-β-Gal staining after immunofluorescence imaging as the fixation and staining may alter antigenicity. Count at least 200 cells from multiple fields for statistical significance. |
| Batch-to-Batch Consistency | X-Gal content within ±5% of specification; Staining Solution pH 6.0 ± 0.1; staining performance verified in senescent vs. proliferating cell controls. |
For research use only, not for clinical use.
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