One of the most common assays run on our FACSCalibur is cell cycle using propidium iodide (PI) as a dye. PI binds stoichiometically to DNA and can reflect the amounts of DNA present within the cell nucleus. The cell cycle is then determined by the strength of the signal obtained from single nuclei as they pass through the cytometer and is often presented as a histogram with three areas of interest: G1 the left hand peak, G2 the right hand peak and S-phase, the plateau seen between G1 and G2 (left hand image from CRUK site).
We can then ustilise analysis programs such as Modfit or FlowJo to analyse the data and give us the % of cells in G1, G2 and S (right hand image from FlowJo web page).
This is great but we can get more information. For instance, are the cells in the bottom right of the G1 peak late G1 or early S-phase? Similarly, are the cells in the bottom left corner of the G2 peak late S or early G2? We need a way of resolving these questions and the most commonly used method is to utilise compounds which will be taken up by cells synthesising DNA (S-phase), and then labelling for this compound. Traditionally bromodeoxyuridine (BrdU) hasbeen used for this purpose. However, accessing the BrdU antigen has often been a technical challenge so a new compound is now available sold by Invitrogen called Click-It Edu. The premise is the same for both. We label calls with BrdU or Edu and then fix and process them. This time
though we utilise a dot plot to show our cell cycle stages with PI across the x axis and e.g. FITC or Alexa488 on the y-axis. The resultant plot then looks a bit like the next figure (again from CRUK flow pages):
As the s-phase cells are now showing positive for the BrdU label they appear bright on the y-axis and can be separated from theG1 and G2 cell stages.
This is a really effective method of improving the information we can obtain from our cell cycle analysis. We can even use this to determine proliferation of cell populatons if we take samples at specified times following a pulse of BrdU or Edu.
This is shown in greater detail on the CRUK flow page http://science.cancerresearchuk.org/sci/facs/facs_major_apps/cell_cycle_analysis/?version=1
Another useful web resource for cell cycle FACS is http://sciencepark.mdanderson.org/fcores/flow/files/DNA_PI.html
No comments:
Post a Comment