11/23/2023 0 Comments Compensation flowjoIf compensation is incorrect, it is simple to generate a new compensation matrix to apply to the samples. Alongside that check-box are options to choose weights per fluorescence detector and to optimize the weighting used. However if clogging is a recurring problem, you may consider filtering your samples prior to running them. In FlowJo v10.6 there is now a check-box labelled Spectral at the top of the compensation wizard to enable spectral compensation functionality. This way you can see the difference before/after compensation. If you are using FlowJo, you can use the export function to create new fcs files where all slashes are replaced with underscores. FlowJo may also try to load an unstained control for use as a universal negative. Checking the “Overlay Uncompensated” checkbox will paint a second layer on each plot that is the uncompensated parameters. FlowJo will automatically try to match the compensation controls to the appropriate detectors, apply a size gate to each of the controls, then try to gate the positive and negative populations in each control.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. With the proper controls, the matrix creation process should be. The workflow was constructed in this manner to discourage users from editing spillover values while creating the matrix. The color coding is a simple heat map – blues are negative spills, and the increasingly darker shades of yellow are applied to higher values. CHAPTER 3 The Matrix Editor The process of creating a compensation matrix and the process of editing a compensation are considered two separate processes in version 10.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.
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