![]() This way you can see the difference before/after compensation. addition of other reagents that were then added and incubated for 20. Checking the “Overlay Uncompensated” checkbox will paint a second layer on each plot that is the uncompensated parameters.Note the heatmap from the matrix is retained here as background of each plot. This view will show current sample’s fluorescent parameters in a grid of either histograms (ALLBY) or NxN bivariate plots.You can use the heatmap to quickly identify the highest/lowest spills of your matrix, which can be useful to track down compensation problems. The color coding is a simple heat map – blues are negative spills, and the increasingly darker shades of yellow are applied to higher values.Modifier keys can be used to accelerate the change – hold down, or, or for finer/coarser changes. You can click inside a field and use up/down arrows to change the numbers. The numbers correspond to the spill values between two parameters.You can uncheck some parameters and then use the “show all” checkbox on top to hide unchecked parameters. The checkboxes on the left control which parameters are being used in the display portion (section 3 below).The provides the capability to apply the current selected matrix to a sample or group by dragging and dropping.The Edit button will duplicate the selected matrix and allow you to edit the compensation spillover values.This can make or break an experimental analysis that spans multiple runs, we recommend using a strict naming convention for your matrices to aid in identifying which data set they belong to. Applying Compensation Matrix in FlowJo FlowJo Media 5.26K subscribers 21 11K views 3 years ago FlowJo Tips Create new, apply old, or compare different matrices. The name field tells you which matrix is currently being edited.Since this window is complex, let’s break it up into 3 pieces and discuss them each below: The matrix editor window can be accessed in two ways:ġ) by double-clicking the compensation badge from the workspace window (any compensated sample has this badge next to the sample in the left hand column down the workspace.)Ģ) by clicking the “View Matrix…” button in the main Compensation window. When you modify the matrix, the numbers are converted to give an estimation of the amount (percentage) of spillover into the other parameter.Starting with version 10 of FlowJo, there is a new interface for viewing compensation matrices – the Matrix Editor. In version 4.5 and after, the matrix can be modified right in FlowJo using the "Edit/Save Matrix" menu option. In very rough terms, the 0.1803 corresponds to approximately 18% spillover (PE-%FITC).Ĭlick here for instructions on modifying the matrix. addition to single-colour controls on beads to facilitate AF correction for manual. Thus, 0.1803 is the spillover coefficient between FITC and PE, or the amount of FITC signal in the PE channel for a FITC-stained cell 0.0091 is the amount of the PE signal in the FITC channel for a PE-stained cell. FlowJo V10.7.1 to create the autofluorescent-corrected compensation matrix. The rows are the spectra of each channel the columns are the contributions of each fluor into the channels. ![]() The subsequent lines are the spillover coefficients, corresponding to each of the channels named. The names must match exactly use the names that appear in the $PnN keywords of the FCS file. Channels that are not in this list are not compensated channels in this list that are not in the sample are ignored. The second line has the names of the fluorescence channels that are being compensated. The first line has the title of the compensation matrix. If you save the matrix to a file (using the "Edit/Save Matrix" option), the file looks like this: CompMatrix When the "Compute" button in the compensation matrix dialog window is clicked, FlowJo generates a compensation matrix.
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