- Cart 0
- English
Cell Cycle Assay Kit: Technical Principles and Experimental Application Guide
February 09, 2026
Clicks：79
Cell cycle regulation and apoptosis are central themes in oncology, pharmacology, and cell biology research. Accurate analysis of cell cycle distribution not only facilitates understanding of cellular proliferation mechanisms but also serves as a critical tool for evaluating drug intervention efficacy and elucidating gene function. Propidium Iodide (PI)-based cell cycle assay kits, characterized by their operational simplicity and intuitive results, have become routine tools in flow cytometry analysis.
What is a Cell Cycle Assay Kit?
A cell cycle assay kit is a reagent combination designed for quantitative analysis of cell population distribution across different cell cycle phases (G0/G1 phase, S phase, G2/M phase). The kit typically comprises three core components: PI staining solution (20× concentrate), RNase A solution (50× concentrate), and staining buffer. PI is a fluorescent dye that intercalates into double-stranded DNA, with fluorescence intensity directly proportional to DNA content. Through flow cytometric detection of fluorescent signals, cell subpopulations with varying DNA content can be clearly distinguished.What is the Technical Principle?
This detection method is based on the regular changes in DNA content during cell cycle progression. In a normal cell cycle, cells in G0 and G1 phases contain 2N DNA content; G2 and M phase cells have completed DNA replication and contain 4N DNA content; while S phase cells exhibit intermediate DNA content between 2N and 4N. After PI intercalates into DNA double strands, it emits red fluorescence upon excitation at 488 nm wavelength, with fluorescence intensity directly reflecting the relative DNA content within cells.
Detection of apoptosis represents an important derivative application of this kit. During apoptosis, the cell nucleus undergoes condensation, and DNA is cleaved into fragments by endonucleases. Following fixation and permeabilization, small DNA fragments leak out of the cell, resulting in fluorescence intensity below the 2N level after PI staining, forming a characteristic "Sub-G1 peak" in flow cytometry histograms, also known as the apoptotic peak. Additionally, apoptotic cells exhibit significantly reduced forward scatter (FSC) signals, and this physical parameter change can corroborate with fluorescence signals.
Experimental Applications
Drug Screening and Pharmacological Evaluation: Rapid detection of compound effects on cell cycle during early drug discovery stages to identify lead compounds with cell cycle regulatory potential.
Gene Function Studies: Observation of cell cycle distribution changes following interference with specific gene expression via siRNA or CRISPR technology to infer gene function in proliferation regulation.
Tissue Sample Analysis: After digestion of solid tissue into single-cell suspensions, assessment of cell cycle status in different cell subpopulations within tissues; note that tissue digestion may affect cell membrane integrity.
Stem Cell Research: Detection of proliferative activity changes during stem cell differentiation or evaluation of culture condition effects on stem cell cycle status.
Detailed Experimental Protocol
Sample Preparation: Adherent cells require trypsin digestion to prepare single-cell suspensions; suspension cells are collected directly by centrifugation. Tissue samples require mincing followed by trypsin digestion and filtration through 200-400 mesh screens to obtain single cells. A critical step is retaining approximately 50 μL supernatant before fixation and vortexing to disperse cell clumps.
Cell Fixation: Use -20°C pre-cooled 75% ethanol as fixative; fix at 4°C for at least 2 hours or overnight. Ethanol fixation also serves a permeabilization function, allowing PI dye to enter the nucleus. Fixed cells can be stored at 4°C for several weeks without affecting detection results.
Staining Working Solution Preparation: Add 25 μL PI staining solution and 10 μL RNase A to every 0.5 mL staining buffer. RNase A functions to digest RNA, preventing interference from RNA-PI binding. This working solution should be prepared fresh and protected from light.
Staining and Detection: Add 0.5 mL staining working solution to each sample and incubate at 37°C protected from light for 30 minutes. Optimal results are obtained when samples are analyzed by flow cytometry within 5 hours after staining, with excitation at 488 nm and detection in the red fluorescence channel. Cell count per sample should be controlled within 1×10⁶ cells; excessive density affects staining uniformity.
Ensuring Experimental Accuracy
Cell Status Control: Cells should be in logarithmic growth phase before experimentation; excessive density leads to growth inhibition and affects the authenticity of cycle distribution. Recommended cell confluency is 70%-80% at the time of treatment.
Optimization of Fixation Conditions: Ethanol concentration and fixation temperature must strictly follow standards. Insufficient fixation leads to DNA loss, while over-fixation may increase background signals. Post-fixation centrifugation speed should not be excessive (recommended 1000 rpm) to prevent loss of fragile apoptotic cells.
Adequate RNase A Activity: Incomplete RNA removal results in additional PI binding, causing broadening of the G0/G1 peak and increased coefficient of variation. Ensure adequate RNase A activity; extend digestion time if necessary.
Light Protection for PI: PI is photosensitive; prolonged light exposure causes fluorescence quenching. The entire process from staining to flow cytometry detection must be strictly protected from light, using aluminum foil to wrap centrifuge tubes or operating in a darkroom.
Standardized Data Acquisition: During flow cytometry detection, appropriate voltage and threshold settings must be established to ensure the G0/G1 peak is positioned appropriately in the linear amplification channel. Standard microspheres are recommended for instrument calibration, and consistent parameter settings across different experiments facilitate result comparison.
Result Interpretation and Common Issues
In a typical cycle distribution histogram, the G0/G1 phase appears as a sharp main peak at 2N fluorescence intensity, the G2/M phase is located at the 4N position, and the S phase forms a diffuse distribution between them. Apoptotic cells appear as a Sub-G1 peak to the left of 2N.
If the G0/G1 peak CV value is excessively large, this indicates suboptimal sample preparation, insufficient RNase A activity, or cellular heterogeneity. If the S phase proportion is abnormally elevated, consider the presence of polyploid cells or abnormal DNA replication. When no obvious Sub-G1 peak is observed but morphological evidence of apoptosis exists, this may result from DNA fragments being too small and completely lost, or inappropriate fixation conditions.
Conclusion
PI-based cell cycle assay kits provide researchers with standardized protocols for cycle analysis. Although this technique cannot distinguish between G0 and G1 phases or G2 and M phases, and requires careful attention to operational details, its high-throughput and quantitative accuracy advantages make it an indispensable tool for cell proliferation research. With the popularization of flow cytometry, mastering the principles and optimization strategies of this technique is of great significance for obtaining high-quality experimental data.
Absin Cell Cycle Assay Kit Recommendation
| Catalog Number | Product Name | Specification |
|---|---|---|
| abs50005 | Cell Cycle Assay Kit | 50 Tests |
Contact Absin
Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.
| Absin Bioscience Inc. worldwide@absin.cn |
 Follow us on Facebook: Absin Bio |