| The cell cycle describes the process of the replication and division of
chromosomes within the nucleus, which occurs prior to cell division. Cancer
cells develop when the normal mechanisms for regulating cell cycle are
disrupted. It is important to identify the genetic basis for this disruption and to
develop therapies to preferentially target those cells with abnormalities. One of
the ways to screen for potentially therapeutic drugs, or the effects of specific
genes on cell cycle, is to measure changes in cell cycle kinetics under varying
conditions. For cells to divide they must first duplicate their nuclear DNA. By
labeling cellular DNA with propidium iodide (PI) you can discriminate cells in
different stages of the cell cycle. Resting cells (G0/G1phase) contain two copies
of each chromosome. As cells progress toward mitosis, they synthesize DNA (S
phase), allowing more PI intercalation with a resulting increase in fluorescence
intensity. When all chromosomes have replicated and the DNA content has
doubled (G2/M phase), the cells fluoresce with twice the intensity of the G0/G1
population. The G2/M cells eventually divide into two cells. Cells can be fixed,
permeabilized and stained with PI according to the protocol below. Data from the
stained cells are acquired on the Guava® PCA™, Guava PCA-96, Guava PCA-96
AFP, Guava EasyCyte™, or Guava EasyCyte Mini using CytoSoft™ software.
Data are displayed in a single parameter histogram. Four markers are available to
analyze the various populations including the optional fourth marker to discern
apoptotic cells, cell aggregates, or an internal standard. Statistics for each
population within the histogram in CytoSoft include percentage of total, and PM2
mean, median, and %CV of fluorescence intensity. |
