Here is a collection of external links about Compensation.Īll of the pages linked above, also link back here for your convenience. 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. The Matrix editor – used to view/adjust matrix numbers with a preview of the changes.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. Are you new to data analysis in FlowJo or looking for a starting point This video will give you a quick overview on compensating your flow cytometry data in. Therefore, in multi-color flow cytometry, each channel will detect the primary fluorescence, but also secondary fluorescence arising from other overlapping fluorophores. 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. It is recommended to leave this value at zero unless these digitization errors become apparent.Compensation has undergone a major redesign for FlowJo Version 10!Ĭompensation in flow cytometry is the process of correcting for fluorescence spillover emissions. This will add a small random value to the linear value prior to it being reconverted back to log. Typically, this value should be less than one. However, if you notice digitization errors, you can enter a value to spread the grouped population out using the Range of random value to add when converting log data field (Figure 9.11). The conversion to linear and reconversion back to log occurs automatically during the software compensation process. Compensations can be edited directly from the Compensation Matrix tab and Spillover Matrix tab by clicking on the Target (Matrix Editor) (Figure 9.9, left) or. Depending on the resolution of your original file, this can result in digitization errors, which can cause large numbers of events to group in a single channel. It is then converted back to log for display. If you have stored data on a logarithmic scale in your FCS file (common with FCS 2.0 data files), this data has to be converted to a linear scale before compensation is applied. You may find it helpful to enter the date or experiment number as the compensation name.Ĭompensation must be performed on linear data. The name of your compensation may be changed at any time by selecting the compensation and clicking the Rename selected compensation button or by right-clicking on a compensation and choosing Rename. When a compensation value is manually adjusted, the compensation icon (red box at the top) becomes red highlighted, while the text value (red box at the bottom) becomes bold and underlined.Įvery compensation must be given a unique name.
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