Ensure that the cytometer is performing within specifications using standard beads.We always recommend reviewing the flow cytometer manufacturer's instructions for detailed compensation guidelines. However, the following guidelines should be suitable in most cases. The procedure for setting correct fluorescence compensation is essentially the same on any cytometer but there are differences between the various available instruments, which makes it difficult to provide a "one size fits all" protocol. (PE-Cy5 + PE overlap) - (PE overlap) = accurate PE-Cy5 results General procedure Adjust the compensation settings until no PE signal is seen in the PE-Cy5 channel (see the procedure below).Observe the signal in both PE and PE-Cy5 channels. Run a sample stained only with a PE-labeled antibody.Excitation and emission spectral profiles This will be seen as "false positive" signals in the PE-Cy5 channel and fluorescence compensation is needed to correct for this overlap. In the example below, following excitation with 488 nm light, PE emission is largely detected in the detector specific for PE but the emission tail lies within the range of the bandpass filter used for detection of PE-Cy5. This ensures that the fluorescence detected in a particular detector derives from the fluorochrome that is being measured. To correct for this spectral overlap, a process of fluorescence compensation is used. However, when emission spectra overlap, fluorescence from more than one fluorochrome may be detected. Within a flow cytometer, the appropriate ranges of excitation and emission wavelengths are selected by bandpass filters. The excitation spectrum is a range of light wavelengths that add energy to a fluorochrome, causing it to emit light in another range of wavelengths, the emission spectrum. What is compensation?Īll fluorochromes have excitation and emission spectra.
#Compensation in flowjo manual#
Please refer to the manufacturer's instructions and software manual for a more detailed compensation procedure for your instrument.
#Compensation in flowjo how to#
The presence of HLA-B27 antigen is strongly associated with ankylosing spondylitis (AS), a chronic inflammatory disease of the axial musculoskeletal system, and a few other rheumatic disorders (Reiter’s syndrome, acute anterior uveitis, and inflammatory bowel disease).This article describes why compensation is required for flow cytometry and how to apply it. Take advantage of the protocols available for various applications, such as lymphocyte and stem-cell enumeration, HLA immunophenotyping and more.īD FACSCanto™ Clinical software is an IVD product for enumerating lymphocyte subsets of samples stained with BD Multitest™ 4-Color and 6-Color reagents.Ī BD ® Stem Cell Enumeration module for BD FACSCanto™ Clinical Software offers a validated analysis software, designed to meet productivity and quality requirements of clinical laboratories for the BD ® Stem Cell Enumeration Kit.Ī BD ® HLA-B27 Application module for the BD FACSCanto™ II System provides a complete system for rapid detection of HLA-B27. Example of result dot plots for a fresh cord blood specimen following acquisition and analysis with the BD ® Stem Cell Enumeration module in BD FACSCanto™ Clinical Software.
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