Flow Cytometry

Flow Cytometry Compensation and Antibody Fluorescent Labels

The inherent overlap of emission spectra of antibody fluorescent labels makes compensation necessary. Compensation is the process by which each fluorescent PMT can substract a percentage of the signal from other PMT’s (PMT detectors). Significant spill-over of fluorescent light may occur, The best way for determining the fluorescence compensation requires running samples that are individually stained with the fluorochrome components of your multi-color samples (5). Compensation controls: In experiments, which require simultaneous staining using two or more fluorochromes, it is necessary to prepare controls that have been stained with each dye separately. These controls, termed compensation controls, are required for adjusting cross-over signals between dyes or detectors. This is critical for multicolor immunophenotyping. Statistically, you set the position of regions so that you separate your positives from your negatives. Look at the Mean peak position corresponding to the appropriate region for the parameter you are compensating. They should be the same (4,5). For the autofluorescence correction, an unstained control sample is used to measure the autofluorescence signals within each fluorescence channel. The natural fluorescence of each cell cluster in an unstained control sample has to be subtracted from the observed fluorescence of the respective cell cluster in the stained sample.

References

1. Janeway, C.A., Travers, P., Walport, M., and Capra, J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA

2. Delves, P. and Roitt, I. 1999. Encyclopedia of Immunology. Academic Press Inc., 2nd ed., San Diego, USA

3. 1994. Current Protocols in Molecular Biology. Volume 2. John Wiley & Sons Inc., USA

4. Cruse, J. and Lewis R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA

5. http://biology.berkeley.edu/crl/flowcytometry.shtml

6. Lodish, H., Baltimore, D., Berk, A., Zipursky, Matsudaira, and Darnell, J. 2000. Molecular Cell Biology. American Scientific Books, USA

7. www.spectracell.com/jana.html

8. Andrade, W., Johnston, M. and Hay, J.B. 1998. The relationship of blood lymphocytes to the recirclating lymphocyte pool. Blood 91: 1653-1661.