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| Product Name | PI/RNase Cell Cycle Staining Buffer Kit for Flow Cytometry |
| Catalog No. | CCAT-HMM-0059 |
| Description | A ready-to-use propidium iodide (PI) and RNase staining buffer kit for quantitative cell cycle analysis by flow cytometry. The optimized formulation combines PI for stoichiometric DNA staining with DNase-free RNase A to eliminate RNA interference, along with sodium citrate and Triton X-100 for nuclear isolation and permeabilization. This kit produces consistent CVs below 3% for G0/G1 peaks on diploid cells, enabling accurate cell cycle phase distribution analysis. |
| Intended Use | Quantitative cell cycle phase distribution analysis (G0/G1, S, G2/M) by flow cytometry; DNA ploidy determination; cell synchronization verification; anti-cancer drug mechanism studies affecting cell cycle progression; apoptosis-associated sub-G1 peak detection. |
| Principle / Technology | Propidium iodide intercalates stoichiometrically into double-stranded DNA; RNase treatment eliminates RNA that would also bind PI; fluorescence intensity of each nucleus is proportional to its DNA content, enabling discrimination of cell cycle phases |
| Detection Method | Flow cytometry; excitation 488 nm (blue laser), emission 585/42 nm bandpass filter (FL2 or PE channel); use linear amplification for DNA content analysis |
| Sample Type | Single cell suspension from cultured cells, dissociated tissue, or bodily fluids; requires 1 × 10^6 cells per sample; fixable for batch processing |
| Sensitivity / LOD | CV <3% for G0/G1 peak on diploid human lymphocytes; resolution enables detection of aneuploid populations with DNA index difference >5% |
| Specificity | Stoichiometric DNA binding; RNA interference eliminated by RNase A treatment; PI also stains dead cells (increased uptake) — gate out debris by forward/side scatter |
| Reaction Conditions / Protocol | Harvest cells; wash with cold PBS; fix with 70% cold ethanol (dropwise while vortexing); incubate at -20 °C for minimum 2 hours or overnight; wash twice with PBS; resuspend in 500 µL PI/RNase staining buffer; incubate 30 minutes at room temperature in dark; filter through 35 µm mesh; acquire on flow cytometer at low flow rate |
| Components / Formulation | Propidium iodide solution (1 mg/mL in PBS), DNase-free RNase A (10 mg/mL), staining buffer (sodium citrate with Triton X-100, pH 7.4) |
| Storage Conditions | Store at 2–8 °C; protect PI from light; stable at this temperature |
| Shelf Life | 12 months from manufacture date; working staining solution prepare fresh or use within 24 hours at 4 °C |
| Package Specifications | 100 tests, 200 tests, 500 tests; test defined as 1 × 10^6 cells per sample |
| Product Form | Liquid reagents, ready-to-use after mixing |
| Key Features | Optimized combined PI/RNase staining buffer; consistent low CV for diploid peaks; includes detailed cell cycle analysis protocol; compatible with ethanol-fixed samples for batch processing; Triton X-100 permeabilization eliminates need for separate permeabilization step; validated on BD, Beckman Coulter, and Miltenyi flow cytometers |
| Purity | PI purity ≥95% by HPLC; RNase A free of DNase activity (tested by plasmid digestion assay); endotoxin <0.1 EU/mg RNase |
| Concentration | As specified per kit; sufficient for stated number of tests |
| Activity / Unit Definition | Apoptosis induction confirmed by positive control treatment producing ≥5-fold signal increase |
| Molecular Weight | Varies by dye and enzyme component |
| Source / Origin | Recombinant annexin proteins; synthetic fluorophore conjugates; chromogenic/fluorogenic peptide substrates |
| pH Range / Optimal pH | Binding/washing buffer pH 7.2–7.4 |
| Shipping Conditions | Cold pack 2–8 °C; protect PI vial from light with aluminum foil during transit |
| Expiration Date / Stability | 12 months at 2–8 °C; PI is light-sensitive — always store and handle in amber vials or wrapped in foil; discard PI if color changes from red to brown/orange indicating degradation |
| Regulatory / Compliance | For laboratory and research use only; RUO; PI is a potential mutagen — handle with gloves; avoid skin contact and inhalation; manufactured under ISO 9001 |
| Compatibility | Compatible with all standard flow cytometers equipped with 488 nm laser; ethanol fixation is critical — formaldehyde or paraformaldehyde fixation causes increased CV; not compatible with cells fixed in methanol |
| Recommended Buffer System | Annexin binding buffer with calcium; cell lysis buffer with protease inhibitors for caspase assays |
| Application Notes / Precautions | For optimal CV, fix cells by adding ice-cold 70% ethanol dropwise to cell pellet while vortexing at medium speed. Always run an unfixed, unstained control for PMT voltage setting. Acquire at low event rate (100–300 events/second) for best CV. Use doublet discrimination (PI-A vs PI-W or PI-H) to gate out aggregates. Analyze with ModFit, FlowJo, or FCS Express cell cycle platforms. |
| Batch-to-Batch Consistency | Positive control signal within ±20% of reference lot; staining pattern consistent |
For research use only, not for clinical use.
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