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Nexcelom Video Presentation

Cellometer Image Cytometry for Cell Cycle Analysis

Cellometer Vision CBA Analysis System

This presentation will focus on using propidium iodide for cell cycle analysis. PI is a membrane exclusion dye and an intercalating agent that stains the cellular genome upon cell fixation.

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Transcript: Cellometer Image Cytometry for Cell Cycle Analysis

  • Welcome! The topic for today's webinar is Cellometer Image Cytometry for Cell Cycle Analysis. I would like to start the talk by describing the importance of cell cycle research

    One major area of research is Oncology. Since cancer cells exhibit abnormal cell division and proliferation rates it is important to understand and study the cell cycle within those cells. Another area of research is pharmacology.

    Specifically, looking at how pharmacological reagents affect cell cycling in cancer cells, as well as examining the potential effects of drug treatment on normal cells. And finally, a lot of research has been conducted on how various molecular and cellular mechanisms affect cell proliferation and growth by examining cell cycling

  • Cell Cycle can be defined as the period between successive divisions of a cell. And in eukaryotic cells the normal cell cycle can be divided into 4 phases
    1. At G1 or the Gap phase the cells are preparing for DNA replication, and the DNA content between cells is relatively same. The diploid number of a cell is the number of chromosomes in the cell, which is commonly abbreviated as 2n.
    2. Replication of the cellular genome occurs during the S phase. Because the replication of the DNA from cell to cell is not synchronized, there is a broad distribution of DNA content between the cells. The DNA content at this phase is between 2n and 4n. By the end of the Synthesis phase the DNA content within the cell is doubled
    3. The third phase of the cell cycle is the G2 phase. At this phase the DNA replication is now complete, and the cell is growing and preparing for cell division. Since the DNA replication is complete, the cells contain twice the amount of the DNA found at G1.
    4. Finally, cell division occurs during the Mitosis phase. At the end of mitosis, both the daughter and mother cells again contain the same amount of genetic material as in the G1 phase.

  • There are multiple methods and protocols for cell cycle detection.

  • One method is by measuring the incorporation of radio-labeled thymidine into dividing cells. The amount of incorporated thymidine can be measured with a scintillation beta-counter. Although many seminal cell cycle studies have been performed using this technique, the hazardous nature of these compounds has led to the development of new and more preferred fluorescence-based methods.
    Fluorescent-based detection methods can be performed on live or fixed cells. Because Hoechst 33342 is cell permeable, it is able to bind to the double-stranded DNA in live cells.

  • There are multiple fluorescent-based detection methods for fixed cells.
    One is using BrdU(Bromodeoxyuridine). BrdU is a thymidine analog and is incorporated into the genome during the S-phase of the cell cycle. Fluorescently –labeled anti-BrdU antibodies are then used to detect the incorporated BrdU.
    Another is DAPI. Because DAPI has poor live cell permeability properties it is most often used post-fixing.

  • Today, I will focus on using propidium iodide for cell cycle analysis. PI is a membrane exclusion dye and an intercalating agent that stains the cellular genome upon cell fixation.

  • Labeling DNA with propidium iodide (PI) allows for fluorescence-based cell cycle analysis. The data generated in this assay is represented as a histogram. The y-axis represents the cell number and the x-axis is the PI-fluorescent intensity. The amount of PI fluorescent intensity is correlated to the amount of DNA within each cell.

    As the cell progresses through the cell cycle, the amount of DNA within the cell changes from 2n at G1 to 4n at G2 and therefore the amount of PI that is incorporated into the genome also doubles. This is reflected in the histogram intensity readout:  as the cell progresses through the cell cycle the fluorescent intensity increases from 2000 to 4000 relative fluorescent units.

  • In a typical experiment
    1. Control or treated cells are incubated for 24 hours
    2. The cell are collected
    3. Fixed for 15 minutes
    4. Stained with PI cell cycle reagent for 40 minutes
    5. Re-suspended in a final volume of PBS
    6. And analyzed using the Vision CBA system

  • Once the cell samples are prepared
    1. Load 20 µL of the cells into a counting chamber
    2. Insert the chamber into the Cellometer
    3. Select the cell cycle assay from the drop down menu and analyze.

  • Brightfield and fluorescent images are captured by the Cellometer and can be reviewed to assure that only the cells of interest were counted.  The data is exported to FSC express software and a cell cycle histogram with a data table generated.

  • In this cell cycle experiment cells were treated with different concentrations of Nocodazole, a drug that is designed to arrest the cell cycle at the G2/M Phase.

    The cell cycle arrest in these Jurkat cells occurred in dose dependent manner. The percent of cells that arrested in the G2/M increased from 14.3% in the control sample to 47.8% in the highest dose Nocodazole treated sample.

  • These types of studies have been performed on the Cellometer as well as on a flow cytometer.  We have been able to show an excellent correlation between the Cellometer Vision CBA system and flow cytometry. As the concentration of Nocodazole increases the percent of cells arresting in the G2/M also comparably increases for both the Cellometer and Flow systems.

  • Here are some other examples for which cell cycle analysis has been performed.
    Prostate cancer -PC3 cell, cervical cancer HeLa cells, pancreatic cancer PANC-1 cells, colon cancer HT-29 cells.

  • Cellometer Vision CBA system is also capable of performing UV-based cell cycle analysis using DAPI.
    In this experiment control and Nocodazole treated PC3 cells were fixed, stained with DAPI and analyzed. The drug treated PC3 cells as expected arrested in the G2/M phase of the cell cycle.

  • In conclusion:
    1. The Vision CBA system is a fast and easy to use tool for performing cell cycle analysis.
    2. The Cellometer can be utilized to effectively measure and analyze a large variety of mammalian cells.
    3. There is an excellent correlation between the Cellometer results and flow cytometry results.
    4. The cell cycle histogram read-outs allow for an easy comparison between control and drug treated samples.
    5. There is an excellent correlation between the ellometer results and flow cytometry results.