Cell Cycle Analysis

Cell cycle progression is a tightly regulated process that involves the duplication of nuclear DNA content prior to cell division. The control mechanisms that regulate this process are often disrupted in tumor cells and serve as viable targets for therapeutic compounds in the treatment of cancer. Automated imaging and analysis techniques enable detailed characterization of cell cycle progression and determination of mitotic index within a microplate format.

Stages of the cell cycle and mitosis

Figure 1. Stages of the cell cycle and mitosis

 

Using nuclear staining and automated imaging to determine percentage of a cell population in different phases of the cell cycle

Population-based cell-cycle analysis using quantifiable fluorescent DNA stains is a well-established method to distinguish cells in different phases of the cell cycle. Automated imaging and analysis protocols using either the Agilent BioTek Lionheart Automated Microscope or Agilent BioTek Cytation Cell Imaging Multimode Reader provide a convenient and robust method for conducting cell-cycle analysis. 

  • The fluorescence intensity of stained cells correlates with the amount of DNA they contain.
  • Nuclear stains such as Hoechst 33342 are specific to DNA and provide an effective option for imaging-based methods for cell cycle analysis. 
  • Low magnification imaging (4x objective) and wide field of view camera provide a large representative sample size for analysis. 
  • Compatible with kinetic live-cell formats.

Relationship between cell cycle and DNA content histogram.

Figure 2. (A) Relationship between cell cycle and DNA content histogram. As cells progress through the cell cycle, their DNA content doubles prior to mitosis. (B) Cells treated with the nuclear stain Hoechst 33342 exhibit fluorescence proportional to their DNA content which is quantified using Gen5 image analysis tools and the total object fluorescence metric. 

 

Histogram analysis of nuclear staining from Mevinolin and Nocodazole treated PC-3 cells.

Figure 3. Histogram analysis of nuclear staining from Mevinolin and Nocodazole treated PC-3 cells. (A) Mevinolin and (B) Nocodazole treatments were used as controls to define G1 (RED) and G2 (BLUE) subpopulations. S-phase (GREEN) was defined as the region between G1 and G2.

Determine mitotic index using low mag imaging and marker for mitosis

Mitotic index, which is the percentage of cells undergoing mitosis at any time, is the most common marker used for asynchronous determination of cell cycle length. 


Cell population is imaged at 4x magnification with nuclear marker (Hoechst 33342) and marker for mitosis (antiphospho-histone H3 antibody).

Figure 4. Cell population is imaged at 4x magnification with nuclear marker (Hoechst 33342) and marker for mitosis (antiphospho-histone H3 antibody). (A) Gen5 analysis identifies mitotic cells (yellow mask) relative to total cell counts to report mitotic index which can be used to (B) quantify dose response.

Detailed characterization of mitosis using Gen5 AutoROI module and high-resolution imaging

The Agilent BioTek Gen5 AutoROI module enables users to automatically identify Regions of Interest (ROIs), including diving cells within a population. First, low magnification scans automatically detect and identify mitotic cells using a mitotic marker, followed by automated high magnification imaging of mitotic cells of interest. 

The AutoROI module for Gen5 Image+ and Gen5 Image Prime is a toolset that enables users to automatically identify Regions of Interest (ROIs).

Figure 5. The AutoROI module for Gen5 Image+ and Gen5 Image Prime is a toolset that enables users to automatically identify Regions of Interest (ROIs). Here, low magnification scans automatically identifies mitotic cells using a fluorescent marker for mitosis (antiphospho-histone H3 antibody). Each mitotic cell of interest is then automatically scanned at the desired higher magnification for detailed characterization.

Immunofluorescent images of RPE1 cells in different stages of mitosis.

Figure 6. Immunofluorescent images of RPE1 cells in different stages of mitosis. Fixed RPE1 cells were stained for actin (green), tubulin (red) and DNA (blue). Confocal images representing (A) prophase; (B) anaphase; (C) metaphase; and (D) telophase stages of mitosis.
 

Increase assay throughput and performance

Liquid handling automation
MultiFlo FX

The Agilent BioTek MultiFlo FX or other Liquid Handler can be used to automate the tedious fixing and staining steps required for some cell cycle analysis applications.

Stacking automation:

BioStack

Process and analyze up to 50 plates at a time by integrating Cytation with the Agilent BioTek BioStack Microplate Stacker

 

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For Research Use Only. Not for use in diagnostic procedures.

RA44441.1640625 

 

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