Cell cycle analysis with flow cytometry and propidium iodide

Cell cycle analysis by quantitation of DNA content was one of the earliest applications of flow cytometry.

Flow cytometry with propidium iodide (PI) staining is a widely used technique for analyzing DNA content and assessing cell cycle distribution. This protocol outlines a reliable method for staining fixed or permeabilized cells using PI, a fluorescent dye that binds stoichiometrically to DNA. The procedure enables researchers to distinguish between G0/G1, S, and G2/M phases, the major phases of the cell cycle, based on fluorescence intensity. It is compatible with various cell types and can be adapted for use with other intracellular markers. This method is ideal for high-throughput analysis and is frequently used in cancer research, drug screening, and cell proliferation studies. This protocol is designed for a single time point measurement of cell cycle distribution.

Introduction

PI staining in flow cytometry is a cornerstone technique for evaluating cell cycle progression and DNA content. PI is a DNA fluorochrome and a membrane-impermeant dye that intercalates with double-stranded DNA to stain DNA for cell cycle analysis, emitting red fluorescence upon excitation. As it also binds RNA, RNase treatment is essential to eliminate the background signal. This protocol provides a step-by-step guide for preparing cells, typically as a cell suspension for flow cytometry, fixing and permeabilizing them, and subsequently staining them with a PI staining solution. This method is particularly useful for researchers studying cell proliferation, apoptosis, or DNA damage, as it allows quantification of proliferating cells and detection of cell death resulting from DNA damage or cell cycle arrest. Its simplicity, speed, and compatibility with a wide range of cell types, including whole cells and cell suspensions, make it a go-to approach in basic and applied biosciences. Flow cytometry with PI staining can also be used for cell sorting based on DNA content or surface markers.

Background and principles

The principle behind PI staining lies in its ability to bind DNA in a stoichiometric manner, meaning fluorescence intensity directly correlates with DNA content. This allows for precise discrimination of cells in different phases of the cell cycle. DNA fluorochromes such as PI are used to stain DNA for cell cycle analysis. Cells in the G1 phase exhibit lower fluorescence, while those in S and G2/M phases show progressively higher intensities. Fixation (commonly with ethanol) is required to permeabilize the cell membrane, allowing PI to access nuclear DNA. RNase treatment is crucial to remove RNA, which PI can also bind. This protocol is foundational in cell biology, enabling quantitative analysis of cell populations using flow cytometry.

The DNA of mammalian, yeast, plant, or bacterial cells can be stained by a variety of DNA binding dyes. The premise of these dyes is that they are stoichiometric, that is, they bind in proportion to the amount of DNA present in the cell. Both cell suspensions and whole cells can be used for DNA content analysis, depending on the experimental requirements.

In this way, cells that are in S phase will have more DNA than cells in G1, as cells in the S phase are actively synthesizing DNA, and by the end of S phase, they have double the DNA content compared to cells in G1. So they will take up proportionally more dye and will fluoresce more brightly until they have doubled their DNA content. The cells in G2 (when cells have completed DNA replication) will be approximately twice as bright as cells in G1.

DNA-binding dyes include propidium iodide (PI), 7-aminoactinomycin-D (7-AAD), Hoechst 33342, 33258 and S769121, TO-PRO-3, 4’6’-diamidino-2-phenylindole (DAPI), DRAQ5™ and DRAQ7™, as well as metachromatic dye.

In most cases, cells must be fixed or permeabilized to allow entry of the dye, which is otherwise actively pumped out by living cells. The plasma membrane acts as a barrier to dye entry, so permeabilization is necessary for effective staining. For fixation, alcohol or aldehyde are commonly used. Alcohol is a dehydrating fixative which also permeabilizes. This will allow easy access of the dye to the DNA and gives good profiles (low coefficient of variation, CV). The disadvantage is that it is often incompatible with fluorescent proteins and some surface markers.

If fluorescent proteins or surface markers need to be examined simultaneously, use of an aldehyde (cross-linking) fixative, usually paraformaldehyde is more appropriate. This may lead to poorer quality profiles (higher CVs) but will allow simultaneous detection of other fluorochromes and membrane-bound proteins. However, paraformaldehyde will usually not permeabilize the cell membrane, and so further sample processing is required.

With fixed cells, samples may be accumulated, stained and analyzed at the conclusion of an experiment. Alcohol-fixed cells are stable for several weeks at 4°C. Aldehyde fixed cells are stable for 2 to 3 days.

An alternative method to allow the DNA dye into the cells is to permeabilize them with a detergent. This can be Triton X-100 (0.1%) or NP40 (0.1%). Saponin is not a recommended permeabilizing reagent for DNA analysis as it does not permeabilize the nuclear membrane well. Permeabilized cells cannot be stored for as long as fixed ones and should be processed within hours.

It is also usually necessary to combine a fixation (paraformaldehyde) and permeabilization (Triton X-100 or NP-40) for the intracellular staining. Other methods are also available, e.g. use of citrate buffers (in combination with detergent), although these are not so widespread. There are also some dyes that will enter live cells and quantitatively bind to DNA, these include Hoechst 33342, DRAQ5™ (ab108410) and the DyeCycle dyes.

The method used will depend on the experiment and the information required. For easy setup, with PI staining of DNA content for flow cytometry we recommend our Propidium Iodide Flow Cytometry Kit, which includes a PI staining solution, otherwise, we recommend this protocol.

Stage 1 - STAGE Method

Materials required

• 70% Ethanol
• Propidium iodide (stock solution 50 µg/mL)
• Ribonuclease I (stock 100 µg/mL)

Steps

Harvest the cells in the appropriate manner and wash in PBS. Typically, trypsin is used for adherent cells. If a solution is used to detach cells, centrifuge your cells gently and remove the solution.

Fix in cold 70% ethanol.

• Add drop-wise to the pellet while vortexing. This should ensure fixation of all cells and minimize clumping.

Fix for 30 min at 4°C.

Wash 2 X in PBS.

• Spin at 850 x g in a centrifuge, and be careful to avoid cell loss when discarding the supernatant, especially after spinning out of ethanol.

Treat the cells with ribonuclease.

• Add 50 µL of a 100 µg/mL stock of RNase. This will ensure only DNA, not RNA, is stained.

Add 200 µL PI (from 50 µg/mL stock solution)

Stage 2 - Analysis of results

Steps

Measure the forward scatter (FS) and side scatter (SS) to identify single cells.

Pulse processing can be used to exclude cell doublets from the analysis.

• This can be achieved either by using pulse area vs. pulse width or pulse area vs. pulse height depending on the type of cytometer.

PI has a maximum emission of 605 nm so can be measured with a suitable bandpass filter.

Stage 3 - Expected results

While running the cytometer, the following plots should be displayed:

Steps

Forward and side scatter to identify the cells.

• Pulse shape analysis to identify clumps and doublets (this can be pulse area vs. pulse width or pulse area vs. pulse height depending on cytometer).
• Forward scatter vs. PI signal; PI histogram.

For analysis, first gate on the single cell population using pulse width vs. pulse area.

• Then apply this gate to the scatter plot and gate out obvious debris. Combine the gates and apply to the PI histogram plot.

There are two ways to quantify the percentage of cells in each cell cycle phase:

• By using markers set within the analysis program.
• By using an algorithm that will attempt to fit Gaussian curves to each phase. This is available with some flow cytometry software and is more objective than setting markers by eye.

Comparison to other methods

Compared to other DNA-binding dyes like DAPI, 7-AAD, or Hoechst 33342, PI offers strong fluorescence and reliable quantification of DNA content. Unlike Hoechst dyes, which can penetrate live cells, PI requires fixation, making it unsuitable for live-cell analysis but ideal for fixed-sample workflows. DAPI and Hoechst require excitation by UV light, while PI is excited by visible laser light at 488 nm, making it compatible with standard flow cytometers. In addition to PI, dyes such as FITC or Alexa Fluor 488 emit green fluorescence, which can be detected alongside PI for multiparametric analysis. The detection system uses emission filters to isolate specific fluorescence signals, ensuring accurate measurement of each dye. While DRAQ5 and DyeCycle dyes allow live-cell analysis, they may be more expensive or less stable. PI remains a cost-effective, robust choice for fixed-cell cycle analysis, especially when paired with RNase treatment to enhance specificity.

Applications

PI staining is extensively used in cell cycle analysis, apoptosis detection, and DNA ploidy studies. It enables quantification of proliferating cells and assessment of cell death by identifying apoptotic cells. In cell division studies, PI staining helps demonstrate that after mitosis, a cell divides into two daughter cells. Flow cytometry with PI staining can also be used for cell sorting based on DNA content or surface markers. Normal cells are often used as references for DNA content analysis to ensure accurate ploidy determination. It is particularly valuable in oncology research for assessing tumor cell proliferation and response to chemotherapeutics. The method is also employed in stem cell biology, immunology, and toxicology to evaluate cell health and division. In combination with other markers, PI can be used to simultaneously assess surface antigens and intracellular targets. Abcam’s protocol supports high-throughput screening and is compatible with various cell types, including mammalian, yeast, and plant cells. Its versatility and reliability make it a staple in both academic and industrial research settings.

Cell viability assessment

Cell viability assessment is fundamental in flow cytometric analysis, especially when evaluating cell cycle dynamics and DNA content measurement. In a heterogeneous cell population, distinguishing live cells from dead cells is essential for accurate interpretation of results. Live cells maintain intact plasma membranes, effectively excluding PI and other viability dyes. In contrast, dead cells have compromised membranes, allowing PI to enter and bind to double-stranded DNA, resulting in a strong fluorescent signal detectable by a flow cytometer. By gating out dead cells based on PI fluorescence, researchers can focus on the DNA content and cell cycle status of viable cells, minimizing artifacts and ensuring reliable data. This approach is widely used to assess the impact of experimental treatments, environmental stress, or disease states on cell viability, and is a critical step in protocols involving cell cycle analysis, DNA content, and cytometric analysis of cell populations.

Analyzing fixed cells

Analyzing fixed cells is a cornerstone technique for accurate cell cycle analysis and DNA content measurement. Fixation preserves cellular DNA and stabilizes cell morphology, allowing for subsequent staining with DNA-binding dyes such as PI. The process typically involves permeabilizing the cell membrane, often with ethanol, to enable the dye to access nuclear DNA. This method is particularly advantageous when working with samples that require long-term storage or when live cell analysis is not feasible. The choice of fixative can influence staining quality; for example, ethanol fixation is preferred for DNA content analysis due to its ability to preserve DNA integrity and yield consistent fluorescence intensity in stained cells. Once fixed and stained, cells are analyzed by flow cytometry, which quantifies the fluorescence intensity of DNA-bound dye in individual cells. This provides detailed information on cellular DNA content, enabling precise DNA content analysis, determination of DNA ploidy, and identification of different phases of the cell cycle within the cell population.

DNA ploidy analysis

DNA ploidy analysis is a powerful application of flow cytometry that assesses the number of chromosome sets within a cell, providing critical insights into cell populations and tumor progression. By staining cells with PI and measuring fluorescence intensity, researchers can generate a DNA content frequency histogram that reveals the distribution of cells across different phases of the cell cycle, including G0/G1, S, and G2/M. This analysis also detects apoptotic cells, which appear as a sub-G1 peak due to highly fragmented DNA. DNA ploidy analysis is instrumental in identifying aneuploidy, cells with abnormal DNA content, which is often associated with cancer and other diseases. The ability to rapidly analyze thousands of stained cells enables robust assessment of DNA content, detection of abnormal cell populations, and monitoring of disease progression or therapeutic response. The frequency histogram produced by flow cytometric analysis provides a visual representation of DNA content, supporting both research and clinical diagnostics in oncology and beyond.

Limitations

Despite its utility, PI staining has several limitations. It cannot be used on live cells due to its membrane impermeability, requiring fixation and permeabilization steps that may alter cellular epitopes. PI also binds RNA, necessitating RNase treatment to avoid background fluorescence. The method is incompatible with certain fluorescent proteins and surface markers, especially when alcohol fixation is used. Additionally, fixation can introduce variability in fluorescence intensity if not standardized. PI staining is also limited in multiplexing applications due to spectral overlap with other red-emitting fluorophores. These constraints should be considered when designing experiments involving PI.

Troubleshooting

Common issues with PI staining include high background fluorescence, poor cell cycle resolution, and inconsistent staining. High background often results from inadequate RNase treatment; ensure sufficient incubation time and enzyme concentration. Poor resolution may stem from suboptimal fixation; ethanol fixation typically yields better profiles than aldehyde-based methods. Clumped cells can distort results, so filter samples before analysis. If fluorescence is weak, verify the PI concentration and incubation time. For inconsistent results, standardize cell number and staining conditions across samples. Always include appropriate controls and calibrate the flow cytometer to ensure accurate DNA content measurement.