Lesson 6: Cell Cycle

Beyond immunofluorescence, cell cycle studies are another popular flow cytometetric technique.

The Circle of Life

Research BRCF Flow Cytometry Training Diagram Cell Cycle Circle of Life

Cellular division, or cell cycling, occurs in two distinct phases, Interphase and Mitosis. Interphase consists of the G1, S and G2 phases:

  • G1 – Cell are active and growing. Cells are receptive to signals to begin DNA synthesis
  • S – Cells are actively replicating DNA
  • G2 – Cells are actively preparing for mitosis. Cells contain twice the normal amount of DNA

Mitosis occurs in the M phase in a four-step process (prophasemetaphaseanaphasetelophase) which results in cell division and normal dna content.

G0 is a somewhat controversial stage as some investigators do not believe that it truly exists. Cells in G0 have exited mitosis and are quiescent. These resting cells may be reactivated and enter the G1 phase upon receipt of the appropriate stimuli.

Propidium Iodide Staining

Research BRCF Flow Cytometry Training Cell Cycle Propidium Iodide Staining graphs

Propidium Iodide is a typical cell cycle stain. The dye passes through a permeabilized membrane and intercalates into cellular DNA.The intensity of the PI signal, then, is directly proportional to DNA content. In the above image, we see three different examples of cell cycle analysis. In the first image (upper left), we see a typical cellular state with the majority of cells in the G0/G1 phase (tallest peak). The rightmost peak on the histogram shows the cells in the G2/M state. The area -between these peaks indicates cells within S-phase. The following two histograms show the cells after various drug treatments. The upper-right image shows the cells being arrested in G2. The lower-left histogram shows a marked increase in apoptosis as indicated by the substantial decrease in DNA content (those events showing sub-G0 DNA content). The lower-right histogram shows an overlay of the three histrograms in these states.

As you can probably imagine, it can be very difficult to determine quantitative measurements of each of the cell cycle phases by simple gating-analysis of the cytometric data (Here are some data files if you care to give it a try). A better approach is to use a modelling program. The Flow Core’s FACSCalibur is equipped with Verity Software House’s Modfit LT. This program, and others like it, utilizes peak fitting techniques to automatically model the pi data and provides the desired quantitative data. Each iteration also provides a statistical analysis of the modelling effort to provide an indication of accuracy. A typical such modelling can be seen below.

Research BRCF Flow Cytometry Training Cell Cycle Propidium Iodide Stain Charts

Staining Techniques

For your convenience, we have placed two common Cell Cycle staining protocols on the core web site. The first is a simple ethanol-fixation technique. This protocol produces excellent results and is the most often used amongst flow core clientele. The second, involves a hypotonic lysis of the cell so that free nuclei are analyzed. This procedure can also produce good results and should be considered if you experience problems with the fixation technique.


Research BRCF Flow Cytometry Training BRDU Cell Cycle plots

While PI staining provides a very precise image of the cellular state with regards to cell cycle, it really provides no information regarding the kinetics of cell cycle. BRDU staining is a popular method for beginning to look at the kinetics of the situation. Bromodeoxy-uridine (BRDU) is similar to the DNA precusor, thymidine. During S-phase, in the presence of BRDU, the cells with incorporate BRDU during the S-phase. After a targetted incubation period, fluorescently-labelled antibodies specific to BRDU can be added to provide an indication of which cells have entered the cell cycle during the incubation period. This technique is often used with PI staining in order to provide a clearer image of cell cycle. The image above shows cells labelled with PI and BRDU-Fitc.

Staining Techniques
For your convenience, we have placed a BRDU staining protocol on the core web site. This procedure produces excellent results, but be aware that careful attention must be paid to titrations and incubation times.

Wrapping Up

In order to asses your understanding of the material thus far, please email the answers to the following exercise to the address below. After receiving this I will provide you with access to the next module.

  • We have focussed on Propidium Iodide in this discussion. Do a web search a find two other cell cycle stains. Comment upon their relative benefits or drawbacks compared to PI (viability, CVs, etc).
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