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| Product Name | PI/RNase Staining Buffer for Flow Cytometric Cell Cycle Analysis |
| Catalog No. | CCAT-HMM-0065 |
| Description | Ready-to-use propidium iodide (PI) staining buffer with RNase A for quantitative cell cycle phase distribution analysis by flow cytometry. Propidium iodide, a stoichiometric DNA intercalating fluorochrome, binds to double-stranded nucleic acids in fixed cells with fluorescence intensity proportional to cellular DNA content. Co-formulated DNase-free RNase A digests RNA to eliminate RNA-derived background staining, ensuring that fluorescence signal exclusively reflects DNA content. Cells in G0/G1 phase exhibit a 2N DNA peak, S-phase cells show intermediate fluorescence between 2N and 4N, and G2/M phase cells display a 4N DNA peak suitable for ModFit, FlowJo, or FCS Express cell cycle modeling. |
| Intended Use | Quantitative DNA content analysis by flow cytometry for cell cycle phase distribution determination, ploidy analysis, and detection of aneuploid populations in cancer research, drug mechanism studies, and cell biology. |
| Principle / Technology | PI intercalates between DNA base pairs with fluorescence enhancement >20-fold upon binding (Ex/Em 535/617 nm); fluorescence is stoichiometric with DNA content; RNase A eliminates RNA background; ethanol fixation permeabilizes cells for PI entry. |
| Detection Method | Flow cytometry: 488 nm excitation; PI detection at 585/42 nm (FL2) or >600 nm longpass; linear amplification for DNA content analysis; low flow rate (100-300 events/sec) for optimal CV. |
| Sample Type | Ethanol-fixed cultured cells; fresh and frozen tissue nuclei; alcohol-fixed clinical specimens. |
| Performance Range / Specifications | Applicable to diploid (2N) through aneuploid (>4N) DNA content; linear fluorescence response across 2N-16N DNA content range; optimal G0/G1 peak CV <5%. |
| Sensitivity / LOD | Detection of aneuploid subpopulations representing 2-5% of total cells; DNA index resolution of 0.05-0.10. |
| Specificity | PI binds dsDNA and dsRNA; RNase A treatment eliminates RNA-derived signal for DNA-specific analysis; PI does not differentiate between live and dead cells — fixation standardizes staining. |
| Reaction Conditions / Protocol | Harvest and wash cells; fix in 70% cold ethanol (-20 °C) dropwise while vortexing; incubate ≥2 hours at -20 °C; wash with PBS; resuspend in PI/RNase buffer; incubate 30 min at 37 °C in dark; analyze by flow cytometry. |
| Components / Formulation | Propidium iodide (1 mg/mL in PBS), DNase-free RNase A (10 mg/mL), 10× PBS, detailed protocol. |
| Storage Conditions | Store PI at 2-8 °C protected from light; RNase A at -20 °C; PBS at room temperature. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 100 tests, 500 tests (1×10^6 cells per test). |
| Product Form | PI: red liquid concentrate; RNase A: clear liquid; prepare working solution by mixing. |
| Quality Control | Each lot tested using fixed chicken erythrocyte nuclei (CEN) and human peripheral blood lymphocytes for G0/G1 CV and DNA index accuracy; PI binding stoichiometry verified. |
| Key Features | Ready-to-use staining buffer with RNase; ethanol fixation method preserves cells for batch analysis; PI with high DNA binding affinity; compatible with all flow cytometers; linear fluorescence for accurate DNA content measurement; suitable for paraffin-embedded tissue nuclei. |
| Purity | Propidium iodide ≥95% by HPLC; RNase A DNase-free certified (<0.001% DNase activity). |
| Concentration | PI working concentration: 50 µg/mL; RNase A working concentration: 100 µg/mL. |
| Activity / Unit Definition | RNase A specific activity: ≥70 Kunitz units/mg; PI fluorescence enhancement upon DNA binding: >20-fold. |
| Molecular Weight | Propidium iodide: 668.39 g/mol (C27H34I2N4); RNase A: ~13.7 kDa. |
| Source / Origin | Synthetic propidium iodide; RNase A purified from bovine pancreas. |
| pH Range / Optimal pH | Staining buffer pH 7.4 ± 0.1. |
| Shipping Conditions | Ambient or cold pack; PI is light-sensitive. |
| Expiration Date / Stability | 12 months at recommended storage; PI/RNase working solution stable for 2 weeks at 2-8 °C protected from light. |
| Regulatory / Compliance | For research use only; not for clinical diagnostic procedures. Propidium iodide is a potential mutagen — handle with appropriate PPE. |
| Compatibility | Compatible with ethanol-fixed cells from most mammalian species. Not compatible with methanol-only or formaldehyde-only fixed cells. For Triton X-100 permeabilized cells, use at 4 °C overnight staining protocol. |
| Recommended Buffer System | PBS with 0.1% Triton X-100, pH 7.4. |
| Application Notes / Precautions | Fix cells by adding ice-cold 70% ethanol dropwise while vortexing to prevent clumping. Store fixed cells at -20 °C for up to 2 weeks before staining. Filter cell suspension through 35 µm nylon mesh before acquisition to prevent clogs. Acquire at low event rate (100-300 events/sec) for optimal CV. Use doublet discrimination gating (PI-A vs. PI-W) to exclude aggregates. For DNA ploidy analysis, include normal diploid reference cells. |
| Batch-to-Batch Consistency | PI concentration within ±10% of specification; RNase A activity within ±20% of reference lot; G0/G1 CV <5% in human lymphocyte standard. |
For research use only, not for clinical use.
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