Nexcelom Bioscience
Cell Counting Tutorials

Cellometer Image Cytometry for Cell Cycle Analysis

Cellometer Image Cytometry System

Importance of Cell Cycle Research

Oncology:

Since cancer cells often undergo abnormal cell division and proliferation, it is important to understand the cell cycle of those cells.

Pharmacology:

Studies involving characterization of pharmacological reagents and their effect on cell cycle regulation

Development of drugs to arrest normal cell cycling during chemotherapy treatment to reduce side effects

Cell/Molecular Biology:

To understand molecular and biological mechanisms, it is essential to examine their role in cell cycle.

What is Cell Cycle?

Cell cycle is defined as "the period between successive divisions of a cell." Standard cell cycle of eukaryotic cells is divided into 4 phases.

Cell Cycle

1. G1 (G=Gap) phase. Cells are preparing for DNA replication. At this stage the cells contain about the same amount of DNA. (diploid (2n))

2. S (S=Synthesis) phase. Replication of nuclear DNA occurs at this stage. During S phase there is a broad distribution of DNA content between G1 and G2 phase. The DNA content is doubled by the end of this phase. (2n to 4n)

3. G2 (G=Gap) phase. Replication of the DNA is complete, and the cell is growing and preparing for division. At this stage, the cells contain twice the number of chromosomes (4n) that are found in G1.

4. M (M=Mitosis) phase. Cell division.

Cell Cycle Detection Methods

There are multiple methods and protocols for detecting DNA content during cell cycle:

Radioactivity Measurement

3H-thymidine - Radioactive labeling of thymidine. Upon DNA replication, the radiolabled thymidine is incorporated into new DNA.

Fluorescence-Based Detection

Live cell measurement

Hoechst 33342 - Is cell permeable and therefore do not require cell fixation. Binds to theĀ minor groove of double-stranded DNA.p

Fixed cell measurement

Bromodeoxyuridine (BrdU) - A thymidine analog, is incorporated into the genome during the S-phase of the cell cycle. Detected using anti-BrdU antibodies.

DAPI - Binds to the AT rich regions of the DNA. Poor live cell permeability and therefore often used post-fixing.

Propidium Iodide (PI) - A membrane exclusion dye and an intercalating agent that stains the cellular genome upon cell fixation.

Fluorescence-based Cell Cycle Analysis using PI

Labeling DNA with propidium iodide (PI) allows for fluorescence-based analysis of cell cycle.

Cell Population Histogram
  • This assay generates a cell population histogram (on left) in respect to PI fluorescence intensity.
  • Amount of PI fluorescence intensity is correlated to the amount of DNA within each cell.
  • Since the amount of DNA doubles (2n → 4n) between G1 and G2 phases, the amount of fluorescence intensity of the cell population also doubles.

Cell Cycle Analysis

Cell Cycle Analysis

In this cell cycle experiment, cells were treated with Nocodazole, a compound designed to arrest the cell cycle at the G2M Phase.

Sample Preparation for PI Cell Cycle Analysis

Sample preperation for PI cell cycle analysis

Cellometer Image Cytometry Procedure

Analyze Cells in Just a Few Steps

Cellometer Image Cytometry Procedure

PI Cell Cycle: Jurkat Cells

PI Cell Cycle Jurkat Cell images

Sample Information

  • Cell type: T lymphocyte
  • Disease: acute T cell luekemia
  • Type: non adherent
  • Untreated control
PI Cell Cycle Jurkat Cells graph

Nocodazole-Induced Cell Cycle ArrestĀ 

Nocodazole-Induced Cell Cycle Arrest

Cellometer and Flow Cytometry Correlation

Cellometer and Flow Cytometry Correlation

PI Cell Cycle: PC-3 Cells

PI Cell Cycle: PC-3 Cells images

Sample Information

  • Organ: prostate
  • Cell Type: epithelial
  • Disease: adenocarcinoma
  • Type: adherent
  • Untreated control
PI Cell Cycle: PC-3 Cells

PI Cell Cycle: HeLa Cells

PI Cell Cycle: HeLa Cells images

Sample Information

  • Organ: cervix
  • Cell Type: epithelial
  • Disease: adenocarcinoma
  • Type: adherent
  • Untreated control
PI Cell Cycle: HeLa Cells graph

PI Cell Cycle: PANC-1 Cells

PI Cell Cycle: PANC-1 Cells images

Sample Information

  • Organ: pancreas
  • Cell Type: epithelial
  • Diseases: epithelioid carcinoma
  • Type:adherent
  • Untreated control
PI Cell Cycle: PANC-1 Cells graph

PI Cell Cycle: HT-29

PI Cell Cycle: HT-29 images

Sample Information

  • Organ: colon
  • Cell Type: epithelial
  • Diseases: colorectal adenocarcinoma
  • Type: adherent
  • Untreated control
PI Cell Cycle: HT-29 graph

DAPI as an Alternative Staining Method for Cell Cycle

DAPI as an Alternative Staining Method for Cell Cycle images

Sample Information

  • PC3 cells: Untreated control
  • Organ: prostate
  • Cell Type: epithelial
  • Disease: adenocarcinoma
  • Type: adherent
DAPI as an Alternative Staining Method for Cell Cycle graph

Summary

  • The Cellometer can be utilized to effectively measure and analyze the cell cycle of mammalian cells.
  • There is an excellent correlation between the Cellometer results and flow cytometry results.
  • Cellometer may be used to examine a wide variety of mammalian cells.
Cellometer Image Cytometry System

Viewing: Cellometer Image Cytometry for Cell Cycle Analysis

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