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Lesson 7 - CFSE
Flow Cytometry
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As discussed in lesson 6 , PI and BRDU staining provide detailed information regarding cellular state but very limited information regarding the kinetics of the cell cycle. CFSE provides significantly greater kinetic information.
CFSE - The Cell Division Marker
CarboxyFluoroscein Succinimidyl Ester, or CFSE, is a vital stain which is, generally, not harmful to cells. Upon entering cells, it undergoes esterase cleavage and diffuses throughout the cytoplasm. As cells divide, the CFSE is split equally between the daughter cells resulting in diminished CFSE signal detection. This division, and resultant signal diminishment occurs with each subsequent cell division. This technique is useful both in vivo and in vitro. The in vivo technique is particularly interesting in that it allows for the harvesting and staining of cells with CFSE followed by the injection of treated cells back into the animal. The cells can then be reharvested at a later date and analyzed for CFSE to indicate how many divisions have occurred in a given time frame. Such analysis are often coupled with drug treatment studies to see how treatment affects the frequency of cell division.
In the images above, you can see an example of CFSE analysis via manual gating. Notice in both images, the Initial Population (IP) is the brightest population with each resulting cell division (1-5) showing reduced CFSE signal. Notice also, that the CFSE analysis shown is of a sub-population of cells as identified by PE counterstaining with a selected antibody.
Peak Fitting
In cases where the investigator needs to know not only the number of divisions but also the frequency of cells in each division state, manual gating can be of limited utility due to peak overlap. As with PI and cell cycle, this method tends to produce qualitative rather than quantitative data. When higher accuracy is desired, it is often necessary to use a peak-fitting algorithm. Peak-fitting applies mathematical modeling to the histogram and produces a quantitative analysis, again complete with internal statistical analysis. Many cytometry packages, such as Weasel (with which we are all familiar), FlowJo and Modfit, include peak-fitting routines. The above image shows a Weasel-based analysis. The instructions for this analysis can be found in the Weasel Help file.
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.
- Practice analyzing the CFSE (FL1 axis) with these data files. As with the above example these cells are counterstained in the PE channel (FL2 axis). Choose a data file and tabulate your results. Be sure to include the method of analysis (curve fitting or manual gating), why you chose the method of analysis, the software used for the analysis, and your overall impressions of the technique. If you have trouble with the an analysis, a detailed example can be found here.
Special thanks to Sean Linkes for providing the CFSE data and graphics.
Sean may be reached at the Flow Cytometry Core Main Facility.
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davadams@umich.edu Last updated: March 8, 2005
