![]() The only difference is that these gates, after being added to the In other words, you can drag gates or statistic nodes to a group exactly the same way as you would to another sample. If the current group is All Samples and you delete the sample, then you will be permanently removing the sample from the workspace.Ī group behaves in some ways as a "template sample" for its members. To remove a sample from a group, select the group, then select the sample and press the delete key. Just click on the sample and drag it to the group. You can add samples from the workspace to any group. These criteria should be checked anytime new samples are added to the workspace: if the new samples meet those criteria, they are added to the group (and group-based analyses are automatically performed at once.) ![]() When you create the group, you are given the option of adding samples to the group which fit a set of criteria-this is specified by the Group Definition dialog window. (2) You can create a new group by clicking on the New (1) When you read in a folder of data files, FlowJo creates a group with the name of the folder and automatically adds the samples to the group or ![]() The "All Samples" group can neither be renamed nor deleted. It contains (by definition) all of the samples known to the workspace. There is a special case group: the "All Samples" group. The groups in the upper portion of the workspace window. Any given sample may belong to one or more groups. A group is a collection of samples-and a mechanism by which analyses can be applied uniformly to that collection of samples. To learn more about automatic compensation and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on Mastery Class wait list.Groups are the heart of all the powerful tools in FlowJo. Now if these three rules have been followed, and the compensation doesn’t look right, do not make the mistake of editing the compensation matrix to make the data look better.Īs many as 30% or researchers “tweak” the matrix to make it look better.įollow the above three rules and your compensation will be correct and if you have issues, explore what those problems are and work to resolve them rather than making up fiction by manual compensation. In every case where investigators have had “problems” with compensation, it is because they have violated the above three rules. For example, don’t use Alexa488 to compensate for FITC. The compensation color must be the same as the experimental color. Avoid using the universal negative for compensation.ģ. Background fluorescence should be the same between the negative and positive population. Controls must be at least as bright as the samples they will be applied to. Keys To Automatic Compensationįor automatic compensation to be successful, the following three rules must be followed.ġ. Manual compensation results in overcompensated data, yielding incorrect conclusions. The best practice is to use automatic compensation algorithms that are available in the most current versions of flow cytometry software that are based on the work by Bagwell and Adam (1993) Ann NY Acad Sci 20:167-184, who describe the mathematics behind multiparameter compensation. If you have manually compensated data in your lab notebook–strike it out now. Manual compensation is the process of adjusting the compensation based on how the data visually looks. One of the most common rumors or practices that has been passed down incorrectly by word of mouth over years past is the concept of manual compensation. Spillover is the overlap of a fluorochrome into a second channel due to the physics of fluorescence. This is a mathematical value that ensures that contributions from the fluorochromes not being displayed are not affecting the distribution of the data being displayed. There are a lot of rumors and mysteries that fill laboratory notebooks about the process. Some of these processes are correct while others lead to incorrect compensation, resulting in poor data.Ĭompensation is the process for correcting for the spillover. One of the most important steps in proper flow cytometry is the process of compensation. Written by Tim Bushnell, PhD / Figures courtesy of Pratip K.
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