Cell Cycle Detection Kit, IC-6510
Product Introduction
The cell cycle refers to the entire process that a continuously dividing cell undergoes from the end of one mitosis to the end of the next mitosis. During this process, the cellular genetic material is replicated and doubled, and is equally distributed into two daughter cells at the completion of division. The cell cycle can be divided into interphase and mitotic phase, with interphase commonly subdivided into the quiescent phase (G0), pre-DNA synthesis phase (G1), DNA synthesis phase (S), and post-DNA synthesis phase (G2). The entire cycle can be represented as G1¡úS¡úG2¡úM. DNA content analysis by flow cytometry reflects the distribution of cells across the various phases of the cell cycle, providing a quantitative measure of cellular proliferation status.
This kit utilizes the ability of intracellular DNA to specifically bind to fluorescent dyes such as propidium iodide (PI). As cells in different phases of the cell cycle contain different amounts of DNA, they bind proportionally different amounts of fluorescent dye, resulting in distinct fluorescence intensities detectable by flow cytometry. Cells in G0/G1 phase exhibit a 2N DNA content, S phase cells contain intermediate DNA content between 2N and 4N, and G2/M phase cells possess 4N DNA content.
During apoptosis, cytoplasmic and chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies alter the light scattering properties of cells. In early apoptosis, the forward scatter signal is significantly reduced while the 90¡ã side scatter signal is increased or unchanged. In late apoptosis, both forward and side scatter signals are diminished. Thus, apoptotic cells can be identified by monitoring changes in light scattering properties using flow cytometry. Furthermore, when stained with PI, apoptotic cells exhibit reduced total DNA content, appearing as a hypodiploid population preceding the normal G0/G1 peak. This sub-G1 peak represents cells with fragmented, sub-diploid DNA content and serves as a quantitative measure of apoptosis.
This kit is applicable for DNA content (cell cycle) analysis of cultured cells, including both suspension and adherent cell types.
Product Features
1. Quantitative Cell Cycle Analysis: Propidium iodide stoichiometrically binds DNA, enabling precise discrimination of G0/G1, S, and G2/M phases by flow cytometry with proportional fluorescence intensity.
2. Apoptosis Detection: Sub-G1 peak identification provides simultaneous quantification of apoptotic cells based on reduced, fragmented DNA content.
3. Light Scatter Assessment: Enables complementary evaluation of apoptotic cell populations through changes in forward and side scatter properties.
4. RNase A Included: Eliminates RNA-derived background signal, ensuring DNA-specific fluorescence for accurate cell cycle distribution.
5. Broad Cell Type Compatibility: Suitable for analysis of both suspension and adherent cultured cells from diverse species and cell types.
6. Ready-to-Use Kit Format: Includes PI staining solution, RNase A, and optimized buffer for a complete cell cycle analysis workflow.
Specifications
Size: Available in 20 assays and 50 assays
Detection Method: Flow cytometry (FL2-A or FL3-A channel)
Fluorophore: Propidium Iodide (Ex/Em: 535/617 nm)
Laser Line: 488 nm (blue laser)
Sample Types: Cultured suspension and adherent cells
Parameters Measured: DNA content (cell cycle distribution), sub-G1 population (apoptosis), light scattering properties
Storage and Stability
Storage Conditions: Store all kit components at -20¡ãC, protected from light. PI staining solution is light-sensitive and must be stored protected from light.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed. After thawing, the PI staining solution may be stored at 4¡ãC for up to 1 month protected from light. Avoid repeated freeze-thaw cycles.
Protocol (For Reference Only)
Important: Propidium iodide is a potential mutagen; handle with appropriate safety precautions. Protect PI staining solution from light at all times. For accurate DNA content analysis, acquire samples at low flow rate and gate out doublets.
1. Cell Preparation: Harvest cells by gentle trypsinization (adherent cells) or centrifugation (suspension cells). Collect 1-5¡Á10⁵ cells per sample. Wash cells once with cold PBS and centrifuge at 300 x g for 5 minutes at 4¡ãC. Discard supernatant.
2. Cell Fixation: Resuspend cell pellet in 300 µL of cold PBS, then add 700 µL of ice-cold absolute ethanol dropwise while gently vortexing to achieve a final ethanol concentration of approximately 70%. Incubate at -20¡ãC for at least 2 hours or overnight. Fixed cells may be stored at -20¡ãC for up to 1 week.
3. Cell Washing: Centrifuge fixed cells at 300 x g for 5 minutes at 4¡ãC. Carefully remove ethanol supernatant without disturbing the cell pellet. Wash cells once with 1-2 mL of cold PBS, centrifuge at 300 x g for 5 minutes, and discard supernatant.
4. PI Staining: Resuspend the cell pellet in 500 µL of freshly prepared PI Staining Solution supplemented with RNase A according to the kit instructions. Incubate at 37¡ãC for 30 minutes in the dark, or at room temperature for 30-60 minutes protected from light.
5. Flow Cytometry Analysis: Filter samples through a 40-70 µm cell strainer immediately before acquisition to remove cell aggregates. Analyze by flow cytometry using linear amplification for PI fluorescence (FL2-A or FL3-A). Acquire at least 10,000 single-cell events per sample at a low flow rate. Gate out doublets and debris using pulse-width (FL2-W) versus pulse-area (FL2-A) analysis. Analyze DNA content histograms using flow cytometry analysis software with cell cycle modeling algorithms. Assess apoptotic cell population by quantifying the sub-G1 peak preceding the G0/G1 peak.
Precautions
1. Propidium iodide is a potential mutagen; handle with gloves and appropriate safety precautions. Dispose of PI-containing waste according to institutional guidelines.
2. Protect PI staining solution from light at all times; photobleaching reduces fluorescence intensity and compromises data quality.
3. Ethanol fixation must be performed dropwise with gentle vortexing to minimize cell clumping; aggregated cells cannot be accurately analyzed.
4. Acquire samples at low flow rates to minimize coefficient of variation (CV) of the G0/G1 peak; high CV values compromise S-phase estimation and sub-G1 discrimination.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What is the detection principle of this kit?
A1: The kit utilizes propidium iodide (PI), a fluorescent dye that binds stoichiometrically to double-stranded DNA. Since cells in different cell cycle phases contain different amounts of DNA (G0/G1: 2N, S: 2N-4N, G2/M: 4N), PI fluorescence intensity directly reflects DNA content, enabling cell cycle phase discrimination by flow cytometry. Apoptotic cells with fragmented DNA appear as a hypodiploid sub-G1 peak.
Q2: Why is RNase A included in the staining solution?
A2: Propidium iodide binds to both DNA and RNA. Without RNase A treatment, RNA staining contributes to the total fluorescence signal and broadens DNA content peaks, reducing the accuracy of cell cycle phase discrimination. RNase A digestion ensures DNA-specific fluorescence.
Q3: How does this kit detect apoptosis?
A3: Apoptotic cells are identified by two complementary methods: (1) The sub-G1 peak on DNA content histograms, representing cells with fragmented, reduced DNA content; and (2) Changes in light scattering properties, where early apoptotic cells exhibit decreased forward scatter with maintained or increased side scatter, while late apoptotic cells show decreased signals in both parameters.
Q4: Can I use this kit for live cell cell cycle analysis without fixation?
A4: This kit is designed for fixed cell analysis with ethanol permeabilization. For live cell DNA content analysis, cell-permeant dyes such as Hoechst 33342 are recommended. PI is membrane-impermeant and requires fixation or permeabilization for nuclear access.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research, optimization of cell preparation and staining conditions is recommended for specific cell types and experimental systems.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product. Propidium Iodide is a potential mutagen; review the Safety Data Sheet before handling.
Ordering Information
Catalog Number: IC-6510
Product Name: Cell Cycle Detection Kit
Size: 20 assays / 50 assays
Price:
20 assays: CNY ¥650.00 / USD $65.00 / EUR €78.00 / JPY ¥11700.00
50 assays: CNY ¥1200.00 / USD $120.00 / EUR €144.00 / JPY ¥21600.00
2023 Version
