Protocol for Cell Cycle
Materials Required
Principle
Cell-cycle analysis by flow cytometry measures DNA content in single cells to estimate the fraction of cells in G0/G1, S, and G2/M phases. Propidium iodide intercalates into DNA, and after RNA removal with RNase, fluorescence intensity reflects cellular DNA content: 2N cells are assigned to G0/G1, cells between 2N and 4N to S phase, and 4N cells to G2/M[1][2][3].
DNA-content analysis alone cannot reliably separate G0 from G1 or G2 from M. Ki-67 can distinguish quiescent G0 cells from cycling cells, EdU or BrdU incorporation marks active DNA synthesis in S phase, and phospho-histone H3 staining identifies mitotic cells within the 4N population[4][5][6][7].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
Reagents and chemicals
• Propidium iodide is used as a stoichiometric DNA-binding dye for flow-cytometric DNA-content histograms[1][2][3].• RNase A is used to remove RNA signal because intercalating dyes such as propidium iodide can bind nucleic acids other than DNA[3][8].
• Ethanol or detergent-based permeabilization/fixation is used to allow DNA dye entry into cells for DNA-content measurement[2][3][5].
• EdU is used as a thymidine analog incorporated into newly synthesized DNA during S phase and detected by azide-based click chemistry[6][7][9].
• BrdU is used as a thymidine analog incorporated into newly synthesized DNA during S phase and detected after DNA denaturation by anti-BrdU antibody staining[4][10][11].
Antibodies, probes, dyes, or kits
• Anti-Ki-67 antibody is used to distinguish cycling cells from quiescent G0 cells when combined with DNA-content staining[5].• Anti-BrdU antibody is used to detect BrdU incorporated into newly synthesized DNA[4][10][11].
• Fluorescent azide click-detection reagent is used to detect EdU incorporation without antibody-based DNA denaturation[6][7][9].
• Anti-phospho-histone H3 antibody is used to identify mitotic cells within the G2/M DNA-content population[5][12].
Cells, tissues, isolated organs, organoids, or animals
• Use single-cell suspensions from cultured mammalian cells or dissociated tissue samples; the cited protocols were developed primarily for cultured mammalian cells and flow-cytometric single-cell analysis[4][5][6].Buffers and solutions
• Use phosphate-buffered saline or equivalent isotonic wash buffer for washing cells before fixation and staining, as used in standard flow-cytometric cell-cycle workflows[4][5][6].• Use PI/RNase staining solution for DNA-content analysis; published PI workflows report PI concentrations in the approximate range of 25-100 µg/mL and include RNase treatment when RNA contribution may affect DNA histograms[1][2][3][8].
Equipment and instruments
• Use centrifuge tubes or plates, pipettes, centrifuge, fixation-compatible containers, cell strainer when needed for single-cell suspensions, and a flow cytometer capable of detecting PI fluorescence after 488-nm excitation[1][2][3][4].• Use flow-cytometry software capable of singlet gating, debris exclusion, DNA-content histogram modeling, and bivariate analysis when EdU, BrdU, Ki-67, or phospho-histone H3 is included[3][4][5][13].
Controls
• Include an unstained control, single-color controls for compensation, RNase-treated DNA-stained control, untreated proliferating control, cell-cycle perturbation control when relevant, and no-primary-antibody control for antibody-based Ki-67, BrdU, or phospho-histone H3 staining[4][5][6][12].Experimental Procedure
Preparation Steps
• Culture cells under the experimental condition of interest and harvest them as a single-cell suspension.Avoid comparing samples collected at different confluence or growth states unless those differences are part of the experimental design, because cell-cycle distribution changes with proliferative state[4][5].
• For DNA-content-only analysis, prepare fixed or permeabilized cells for PI/RNase staining.
For S-phase-specific analysis, pulse cells with EdU or BrdU before fixation; BrdU pulse-labeling protocols commonly combine BrdU incorporation with total DNA staining to distinguish G1, S, and G2/M more accurately than DNA content alone[4][6][7][10].
• For G0 analysis, plan Ki-67 plus DNA-content staining.
For mitotic analysis, plan phospho-histone H3 plus DNA-content staining, because these markers resolve cell-cycle states that cannot be separated by DNA content alone[5][12].
Operation Steps
• Harvest cells and generate a single-cell suspension suitable for flow cytometry.Remove clumps before acquisition because aggregates can falsely appear as higher-DNA-content events[3][4][13].
• Fix or permeabilize cells using a published workflow compatible with the intended readout.
Ethanol fixation is widely used for DNA-content and BrdU workflows, while detergent-based rapid permeabilization has been reported for concurrent PI and phospho-histone H3 staining[4][5][12].
• Stain DNA with PI in the presence of RNase.
Published PI DNA-content studies report stable DNA histograms using PI in the 25-100 µg/mL range, and RNase treatment is recommended when RNA-associated fluorescence may distort DNA-content measurement[1][2][3][8].
• For EdU S-phase detection, pulse-label live cells with EdU, fix/permeabilize, and detect incorporated EdU using fluorophore-azide click chemistry.
EdU detection identifies cells undergoing DNA synthesis and avoids the DNA-denaturation step required for BrdU antibody detection[6][7][9].
• For BrdU S-phase detection, pulse-label cells with BrdU, fix cells, denature DNA to expose incorporated BrdU, stain with anti-BrdU antibody, and counterstain total DNA with PI.
Bivariate BrdU/DNA analysis separates active S-phase cells from G1 and G2/M cells more clearly than DNA content alone[4][10][11].
• For G0/G1 distinction, stain fixed/permeabilized cells with anti-Ki-67 and PI; Ki-67-negative 2N cells are interpreted as G0, while Ki-67-positive 2N cells are interpreted as cycling G1[5].
• For G2/M distinction, stain fixed/permeabilized cells with anti-phospho-histone H3 and PI; phospho-histone H3-positive 4N cells are interpreted as mitotic cells, while phospho-histone H3-negative 4N cells are interpreted as G2-enriched cells[5][12].
• Acquire samples on a flow cytometer using linear DNA-fluorescence scaling for DNA-content histograms when modeling G0/G1, S, and G2/M fractions.
Collect enough singlet events for stable histogram fitting and apply the same acquisition settings to all comparable samples[3][4][13].
Data Acquisition and Analysis
• Gate cells sequentially to remove debris, dead or damaged events when applicable, and doublets or aggregates before cell-cycle modeling.Doublet discrimination is essential because two attached G1 cells can mimic one G2/M cell by total DNA fluorescence[3][4][13].
• Quantify G0/G1, S, and G2/M fractions from PI DNA-content histograms using accepted mathematical modeling approaches rather than manual peak inspection alone[3][13].
• For bivariate assays, interpret EdU-positive or BrdU-positive cells as active DNA-synthesis cells, Ki-67-negative 2N cells as G0, and phospho-histone H3-positive 4N cells as mitotic cells[4][5][6][7][12].
• Normalize results as the percentage of total gated singlet cells or as the percentage within a defined experimental subpopulation.
Use biological replicates for statistical comparison and technical replicate tubes or wells only to assess staining and acquisition reproducibility[4][5][13].
Potential Issues and Alternatives
Q1. Problem
The G0/G1 and G2/M peaks are broad.Possible Cause: poor DNA staining, variable permeabilization, or inappropriate dye conditions.
Literature-supported Solution: use validated PI/RNase staining conditions, keep staining conditions consistent across samples, and analyze only singlet events[1][2][3][13].
Q2. Problem
The apparent G2/M fraction is falsely high.Possible Cause: doublets or cell aggregates are being counted as 4N cells.
Literature-supported Solution: use pulse-width, pulse-area, or equivalent doublet-discrimination gating before DNA-content modeling[3][4][13].
Q3. Problem
PI fluorescence does not reflect DNA content accurately.Possible Cause: RNA contributes to PI fluorescence.
Literature-supported Solution: include RNase treatment when using PI-based DNA-content analysis[3][8].
Q4. Problem
S-phase cells are poorly resolved by DNA-content analysis alone.Possible Cause: DNA-content histograms infer S phase mathematically rather than directly detecting DNA synthesis.
Literature-supported Solution: add EdU or BrdU pulse labeling and analyze bivariate nucleotide-incorporation versus DNA-content plots[4][6][7][10].
Q5. Problem
BrdU staining disrupts multiplex antibody detection.Possible Cause: BrdU detection requires DNA denaturation, which can damage protein epitopes.
Literature-supported Solution: use EdU click-chemistry detection when preservation of other epitopes is required[6][7][9].
Q6. Problem
G2 and M cannot be separated.Possible Cause: both populations have 4N DNA content.
Literature-supported Solution: add phospho-histone H3 staining to identify mitotic cells within the 4N population[5][12].
References:
- [1]. Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol. 1975;66(1):188-193. [Content Brief]
- [2]. Fried J, et al. Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data. J Cell Biol. 1976;71(1):172-181. [Content Brief]
- [3]. Darzynkiewicz Z, et al. Analysis of cellular DNA content by flow and laser scanning cytometry. Methods Mol Biol. 2010;676:137-147. [Content Brief]
- [4]. Cecchini MJ, et al. Analysis of cell cycle position in mammalian cells. J Vis Exp. 2012;(59):3491. [Content Brief]
- [5]. Kim KH, et al. Assaying cell cycle status using flow cytometry. Curr Protoc Mol Biol. 2015;111:28.6.1-28.6.11. [Content Brief]
- [6]. Darzynkiewicz Z, et al. Cytometry of DNA replication and RNA synthesis: historical perspective and recent advances based on “click chemistry”. Cytometry A. 2011;79(5):328-337. [Content Brief]
- [7]. Clarke ST, et al. Click chemistry for analysis of cell proliferation in flow cytometry. Curr Protoc Cytom. 2017;82:7.49.1-7.49.30. [Content Brief]
- [8]. Taylor IW, et al. An evaluation of DNA fluorochromes, staining techniques, and analysis for flow cytometry. I. Unperturbed cell populations. J Histochem Cytochem. 1980;28(11):1224-1232. [Content Brief]
- [9]. Cappella P, et al. A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining. Cytometry A. 2008;73(7):626-636. [Content Brief]
- [10]. Terry NH, et al. Flow cytometry after bromodeoxyuridine labeling to measure S and G2+M phase durations plus doubling times in vitro and in vivo. Nat Protoc. 2006;1(2):859-869. [Content Brief]
- [11]. Cappella P, et al. Miniaturizing bromodeoxyuridine incorporation enables the usage of flow cytometry for cell cycle analysis of adherent tissue culture cells for high throughput screening. Cytometry A. 2011;79(1):26-34. [Content Brief]
- [12]. Shen Y, et al. Rapid profiling cell cycle by flow cytometry using concurrent staining of DNA and mitotic markers. Bio Protoc. 2017;7(16):e2517. [Content Brief]
- [13]. Dean PN, et al. Mathematical analysis of DNA distributions derived from flow microfluorometry. J Cell Biol. 1974;60(2):523-527. [Content Brief]
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