Centre Optical Instrumentation Laboratory

Cell Cycle Analysis on FACSCaliber

The Cell Cycle
G1 Phase
The start of cellular reproduction where RNA and protein synthesis occur. The amount of DNA remains
constant (known as 2C DNA), however cells at G0 and G1 both contain 2C DNA and cannot be distinguished
so cells in this phase are known as G0/G1
S Phase
DNA synthesis phase which continues until the DNA content of the cell is doubled and becomes 4C DNA
G2 Phase
Cell is preparing for cell division, DNA content remains 4C
M Phase
Cell divides by mitosis, DNA remains 4C until the cell has completely split into 2 cells

The graph below demonstrates a distribution of cells in the cell cycle from a flow cytometer.
DNA Graph
NOTE. This is only a representation, your results are unlikely to look exactly like this.
Propidium Iodide is one of the more commonly used fluorophores for cell cycle analysis. It can be used to quantitatively assess teh effects of drug treatment or gene transfection on the cell cycle. However, there are numerous other fluorescent dyes which bind stoichiometrically to DNA such as
  • DAPI
  • Hoechst 33342 (will work in live cells)
  • 7 Aminoactonomycin D
  • Propidium Iodide
  • DRAQ5
  • TO-PRO-3
Our flow cytometer has no UV laser so DNA labelling is best accomplished with Propidium Iodide, DRAQ5 or 7 Aminoactonomycin D

Doublets

A doublet is 2 particles passing through the sensing area together being classed as a single event. This artificially increases the amount of DNA content in the G2/M area. However the cytometer is capable of descriminating this event.
This is best achieved by plotting pulse width (FL-3W) vs pulse area (FL-3A). Note the graph is a representation
Doublet
The single events can then be gated to extract the data from the single events. (Graph is a representation
Doublets

Factors Affecting Flow Cytometry Results

  • Sample Preparation - Try and use single cell suspensions with as little cellular debris and aggregates as possible.
  • Cell Concentration - Try to have a final cell concentration greater than 105
  • Staining - Only use fluorescent dyes which bind stoichiometrically and specifically to cellular DNA