It is recommended to leave this value at zero unless these digitization errors become apparent. Once you have a matrix in FlowJo, it will appear as a node similar to other nodes in FlowJo (gates, stats, platforms, etc.): If multiple matrices are required, several compensation groups must be created. Add as many tubes to the experiment as necessary. In addition to automatically organizing your single-stain controls, the compensation group also stores your matrix nodes. Select Import compensation matrix and gain. Select Compensation Matrix in the Settings menu. This will add a small random value to the linear value prior to it being reconverted back to log. If compensation was performed import it (using the compensation icon in the tube window) and apply to all tubes (Import>Compensation). This will start the process of importing the compensation and analysis (the calculated statistics) from a FlowJo. Typically, this value should be less than one. However, if you notice digitization errors, you can enter a value to spread the grouped population out using the Range of random value to add when converting log data field (Figure 9.11). Details about these parameters from the FlowJo docs are. ![]() The conversion to linear and reconversion back to log occurs automatically during the software compensation process. The FlowJo biex transform has similar input parameters, with some subtle but important differences. Depending on the resolution of your original file, this can result in digitization errors, which can cause large numbers of events to group in a single channel. It is then converted back to log for display. If you have stored data on a logarithmic scale in your FCS file (common with FCS 2.0 data files), this data has to be converted to a linear scale before compensation is applied. You may find it helpful to enter the date or experiment number as the compensation name.Ĭompensation must be performed on linear data. The name of your compensation may be changed at any time by selecting the compensation and clicking the Rename selected compensation button or by right-clicking on a compensation and choosing Rename. When a compensation value is manually adjusted, the compensation icon (red box at the top) becomes red highlighted, while the text value (red box at the bottom) becomes bold and underlined.Įvery compensation must be given a unique name. Then, navigate to the Compensation tab, usually located at the top of the workspace. When you modify the matrix, the numbers are converted to give an estimation of the amount (percentage) of spillover into the other parameter.Figure 9.9. Access compensation settings: In FlowJo, select the sample(s) for which you want to import the compensation matrix. 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. The compensation matrix is represented by the grid to the left of the sample name. 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. Applying a compensation matrix Create new, apply old, or compare different matrices. 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. Once you have a fully-filled compensation dialog window as shown left, simply click on the Compute or Compute and Close button.
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