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Review
. 2023 Feb 12;24(4):3674.
doi: 10.3390/ijms24043674.

Basic Methods of Cell Cycle Analysis

Anna Ligasová  1 , Ivo Frydrych  1 , Karel Koberna  1
Affiliations
Review

Basic Methods of Cell Cycle Analysis

Anna Ligasová et al. Int J Mol Sci. .

Abstract

Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases; the length of these particular cell cycle phases is an important characteristic of cell life. The progression of cells through these phases is a highly orchestrated process governed by endogenous and exogenous factors. For the elucidation of the role of these factors, including pathological aspects, various methods have been developed. Among these methods, those focused on the analysis of the duration of distinct cell cycle phases play important role. The main aim of this review is to guide the readers through the basic methods of the determination of cell cycle phases and estimation of their length, with a focus on the effectiveness and reproducibility of the described methods.

Keywords: BrdU; DNA labeling; EdU; cell cycle; labeled nucleosides; markers of cell cycle phases; time lapse microscopy.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Models of the growth curves: (a) exponential growth curve; (b) logistic growth curve. (a) The exponential growth curve was modeled in GraphPad Prism 6 software (Dotmatics, Boston, MA, USA) using Plot a function analysis using the Exponential growth equation with the following parameters: P(0) = 1; k = 0.03 (k was derived from Equation (14) with the doubling time D equal to 24 h). (b) The logistic growth curve was modeled in GraphPad Prism 6 software (Dotmatics, Boston, MA, USA) using Plot a function analysis using the Logistic growth equation with the following parameters: P(0) = 1; k = 0.03 (k was derived from Equation (14) with the doubling time D equal to 24 h; K = 1000, where K is the maximum cell population).
Figure 2
Figure 2
Schema of the cell cycle.
Figure 3
Figure 3
An example of cell cycle analysis using PI. The percentage of cells in distinct cell cycle phases calculated using Kaluza Analysis Software 2.2 (Beckman Coulter, Brea, CA, USA) based on the Michael H. Fox algorithm.
Figure 4
Figure 4
Time lapse analysis of the cell cycle using fluorescently labeled PCNA. Scheme of the cell cycle composed of G1, S, G2, and M phases (not to scale). Duration of particular cell cycle phases were quantified using time lapse fluorescence microscopy by a PCNA-mCherry reporter to identify four discrete events during the lifetime of an individual cell. Fluorescence images were acquired using a Nikon Ti Eclipse inverted microscope with a Nikon Plan Apochromat Lambda 40× objective with a numerical aperture of 0.95 using an Andor Zyla 4.2 sCMOS detector. In addition, the Nikon Perfect Focus System (PFS) was employed in order to maintain focus of live cells throughout the entire acquisition period. The microscope was surrounded by a custom enclosure (Okolabs) in order to maintain a constant temperature (37 °C) and atmosphere (5% CO2). NIS-Elements AR software was used for image acquisition and analysis. Images were acquired every 10 min. Adapted from [59].
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