This way you can see the difference before/after compensation. In the same way, the sample files load into the FlowJo workspace and appear as part of the All Samples group. Select/highlight the Basic Tutorial Data folder, and click Choose. Checking the “Overlay Uncompensated” checkbox will paint a second layer on each plot that is the uncompensated parameters. Using the file browser, navigate to the Basic Tutorial Data folder.Click the Concatenate button at the bottom of the window. Note the heatmap from the matrix is retained here as background of each plot. Expand the Advanced Options section and add a suffix indicating the antibody. This view will show current sample’s fluorescent parameters in a grid of either histograms (ALLBY) or NxN bivariate plots. Advanced Topics Advance your FlowJo knowledge with these advanced tutorials.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. FlowJo video tutorials FlowJo pdf tutorials and datasets, Basic and advanced tutorials and sample data sets. 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.The environment is presented as the Workspace, which contains a list of loaded samples (experimental data), statistics, gates, and other analyses, as well as tabular and graphical layouts. You can uncheck some parameters and then use the “show all” checkbox on top to hide unchecked parameters. Welcome to FlowJo, a software application with an integrated environment for viewing and analyzing flow cytometric data. 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. Please use the navigation bar at left to explore, and. The name field tells you which matrix is currently being edited. Our web manual is a search-able set of pages which explains the many features and functions in FlowJo.The instrument has six lasers and 182 detectors for complete spectral data acquisition, allowing up to +45 color panels, load-and-walkaway data acquisition and advanced autofluorescence subtraction tool. Since this window is complex, let’s break it up into 3 pieces and discuss them each below: Sony ID7000 spectral flow cytometer with advanced high-parameter flow cytometry capabilities. 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. The Clambey laboratory has used this algorithm in publications for the analysis of CyTOF data as well as RNA flow cytometric data.Starting with version 10 of FlowJo, there is a new interface for viewing compensation matrices – the Matrix Editor. Within the VorteX Clustering Environment users can visualize their data by a number of methods (a three dimensional PCA plot, force directed layout, etc.).Īlthough X-shift/VorteX is a very powerful tool for analysis, it does require more advanced computational skills. These iterations are taken into account to determine the optimal number of clusters to prevent the underclustering or overfragmentation of the data via the “ elbow method”. X-shift calculates the impact of different numbers of nearest neighbors on the number of clusters discovered and produces 30+ analysis iterations. When this option is selected FlowJo will create gate boundaries that align with the numerical scale on the axes. Absolute intensity is the scaled intensity value displayed on the axes in FlowJo. The units of range are intensity and percentile. After the graph is constructed the cell event density is used to partition the data into clusters. By default, FlowJo will display the minimum and maximum values. The X-shift algorithm is a clustering algorithm that utilizes the data to construct a weighted k-nearest-neighbor density estimation (kNN-DE) graph.
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