About 5,000 events for both the positive and negative population is ideal, but less can be used if necessary. #Compensation flowjo softwareYou thus cannot use one of them for the sample and the other for the compensation control.Įnough events must be collected for the software to make a statistically significant determination of spillover. The detectors, or channels, in the instrument are designed to. For example, both GFP and FITC emit mostly green photons, but have vastly different emission spectra. Compensation in flow cytometry is the process of correcting for fluorescence spillover emissions. The compensation control must use the same fluorophore as the sample. The entire set of compensation controls may include individual samples of either beads or cells, but the individual samples must have the same carrier particles for the fluorophores. Whether each individual compensation control contains beads, the cells used in the experiment, or even different cells, the control itself must contain particles with the same level of autofluorescence. The compensation algorithm needs to be performed with a positive population and a negative population. Tandem dyes from different vendors or different batches must be treated like separate dyes, and a separate single-stained control should be used for each because the amount of spillover may be different for each of these dyes. Note: Although it would seem safe to assume that all tandem dyes created with the same donor and acceptor would have the same emission, this is not the case. The exceptions to this are tandem dyes, which cannot be substituted. Antibody capture beads can be substituted for cells and one fluorophore conjugated antibody for another, as long as the fluorescence measured is brighter for the control. The staining of the compensation control must be as bright as or brighter than the sample. The important rules to remember when using single stained samples for compensation are: This spectral overlap should be compensated for every fluorophore used. Figure 21b shows how the data looks when properly compensated. Single staining will reveal the level of spectral overlap between different fluorophores and allow you to remove or compensate for this overlap ( Chapter 2, Figure 12). This can be seen in Figure 21a where the fluorescence of FITC can be detected in the PE channel. Here is a collection of external links about Compensation.Īll of the pages linked above, also link back here for your convenience.Single Staining and Compensation ControlsĪs mentioned in Chapter 2 when performing multicolor flow cytometry, single stained samples are essential to determine the levels of compensation. When you are ready to proceed to a more complete overview of the new compensation work flow, please see this document.Īlso, consider the Compensation Tutorial. One other document you will likely find useful is:Īpplying/Renaming – the matrix, a small overview. However, the fluorophores used in flow cytometry do not adhere to the exact range of emission detected by the instrument. The detectors, or channels, in the instrument are designed to detect a very specific range of emissions. The Matrix editor – used to view/adjust matrix numbers with a preview of the changes. Compensation in flow cytometry is the process of correcting for fluorescence spillover emissions.The Compensation window – used to define a compensation matrix from single stain controls.The Compensation group – in the workspace and tied to compensation Preferences.Single stained controls used to compensate in FlowJo will need to be scaled correctly ahead of time.įor a more complete overview of the theory and principles of compensation, please check out this page.įor version 10, there are three main components to the user interface for Compensation: Single stain controls must be run to determine the amount of secondary fluorescence contributed to each channel. Therefore, in multi-color flow cytometry, each channel will detect the primary fluorescence, but also secondary fluorescence arising from other overlapping fluorophores. Is there one better than the other or do they. However, the fluorophores used in flow cytometry do not adhere to the exact range of emission detected by the instrument. For this month FACS Tips, we are adressing the often asked question about compensation in DIVA vs Flowjo. The detectors, or channels, in the instrument are designed to detect a very specific range of emissions. Compensation has undergone a major redesign for FlowJo Version 10!Ĭompensation in flow cytometry is the process of correcting for fluorescence spillover emissions.
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