I also first learned the basics of flow with the FACSCalibur/CellQuest - manual compensation is pretty convenient when you only have four colors, but if you move to an instrument that allows bigger panels, manual comp becomes pretty cumbersome, and it ends up being easier and probably better to compensate using FlowJo. If you want to be sure its working, you could compare the manually-generated compensation to the one generated by FlowJo - it should give you the same answer. Just run a single-color sample for every color in your panel and you'll be able to compensate your experimental samples in FlowJo. CytoRSuite contains tools for every stage of the analysis pipeline including user-guided automatic compensation, realtime editing of spillover matrices, manual gate drawing, gate editing, visualisation of all existing flow cytometry objects and export of population level statistics for further analysis. Thus, for that sample, if there is PE signal, then it must be spillover, and will be subtracted out by the software. An intuitive and interactive approach to flow cytometry analysis in R.
Compensation flowjo software#
The key thing here is that your compensation controls are single-color - so for example if you want to compensate FITC out of the PE channel, you need a "FITC only" control that the software "knows" only contains FITC. Since your single-color compensation controls have to be acquired and analyzed in order to generate the compensation matrix, they are not themselves compensated at the time of acquisition (i.e., you can't apply a matrix that you haven't already generated!). I do this routinely with a 10-color panel. You can swap them simply by selecting them and dragging/dropping the to a sample or group.It will absolutely work to collect your compensation controls and samples without compensation applied and then generate and apply a compensation matrix in FlowJo post-hoc. Thirdly, all your matrices created in the workspace are present in the matrix editor. Secondly, you can always create a new matrix in FlowJo and overwrite the current matrix. If you want to reset it, simply open the matrix editor again and click reset.Ĭompensation matrices never need to be removed!įirst off, you always have access to the uncompensated parameters in the graph window (graph > show uncompensated parameters). I tried to use FlowJo compensation wizard to set up the matrix, but it doesnt recognize the positive population Thats why I turned to the Comp Beads. Click edit to modify the values, give the matrix a new name and use the to apply it to any sample. The acquisition matrix can be modified by double clicking on any compensation matrix badge next to a sample. o If you want to manually fix or adjust the compensation, it can be more difficult o If you want to check that the automatic compensation is correct, it’s more difficult and time consuming to do than in FlowJo o Takes more time on the machine, which you are being charged for NOTE: It is possible to change FACSDiva’s compensation in FlowJo.
When fcs3.0 files are loaded into FlowJo, the acquisition matrix is applied automatically using the $SPILL keyword embedded within the file. Matrix name is derived from the compensation interface while creating the matrix and matrix color is derived from the same location. To open this, click the compensation node UNDER the “Compensation” group.ĭifferent matrices can be distinguished by color and by name. The name of the Matrix is accessible via the main Compensation Window. If you wish to compensate an entire group of samples, drag the compensation node to the group instead. Once you have a Compensation Node created in the compensation editor, drag the from the compensation editor to a sample or a group of samples. If the samples are not added to the compensation group automatically, manually drag them from the “All samples” groups to the “Compensation” group.Īssigning Compensation Matrices to Samples: Consider naming your compensation controls comp- or compensation. The compensation control must be as least as bright as the stained sample. For the most accurate compensation, there are three basic rules that must be followed: 1. Samples will be placed in the “compensation” group if they contain the phrase ‘comp’. 3 Rules for Compensation Compensation uses single stained controls to account for fluorescence spillover and is critical for obtaining good multicolor flow cytometry data. This should happen automatically as you add data. For more details, consider looking at our Compensation Tutorial or Work Flow.Īssigning Control Tubes to the Compensation Group: This page captures some of the work flow gestures in the Compensation user interfaces in FlowJo.
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