This would happen automatically if your files had a acquisition defined compensation matrix associated with them because compensated parameters would exist in the files and FlowJo would automatically apply the gates. This command will commit all of those group’s gates to all of the samples in the group. Once you apply a matrix to your samples, you can right click on any group and select “Unify Analysis”. You can compensate in FlowJo either by creating a new matrix from compensation controls run with these new data (recommended) or you can load a matrix exported from a previous experiment. New Files don’t have an acquisition matrix (uncompensated) Therefore, we need to compensate these new data first. However, if you load data into a template that has not yet been compensated, the gates will not automatically apply because the gates in the template were created on compensated parameters and these new data don’t have any compensated parameters yet. When a template is created on compensated parameters, and new data is loaded that was compensated on the machine (includes an acquisition matrix), these data will load right into the template and gates will be applied. 2013.Applying new compensation matrices to data loaded into templates can be a bit confusing. Quantifying Spillover Spreading for Comparing Instrument Performance and Aiding in Multicolor Panel Design”. Comparison between different instrument configurations – The measurement gives a PI or SRL manager the ability to quantifiably contrast between different cytometers and/or alternate arrangements of a given cytometer.The SSM calculation helps point the way to maximize useful signal. Panel optimization – Immunofluorescence panel design becomes exponentially more complex as researchers utilize increasingly more channels of detection in their experiments.Quality control of instrument sensitivity – Since signal spreading directly effects channel sensitivity and results between similarly configured machines are readily comparable, the SSM can be used as a benchmark of configuration and performance quality control.The values in these matrices are directly comparable, though they only describe one part in a complex system which contributes to the quality of a flow panel. The values within this matrix are not a percentage, but represent a standard deviation of spillover, AKA signal spread. These values can be calculated for each detector staining pair, resulting in the spillover spreading matrix. Here is a graphical example of those measures from Nguyen et. F is fluorescence in units of intensity.Sigma is standard deviation (adjusted to use 84th and 50th percentile).S stands for ‘Stained’, meaning the FL2+ population in this example.R stands for ‘Reference’, meaning the FL2- population in this example.The spillover spreading for a given detector/spillover-color pair can be calculated empirically by taking the square root of differences in squared robust standard deviations of measured fluorescence intensity from unstained negative and singly stained positive control populations: Spillover spreading is construed as the impediment of signal detectable above background, and is intrinsic to the configuration of a given instrument. To generate a spillover spreading matrix in FlowJo visit the compensation editor, select the matrix of interest, and click the “SSM” button: The Calculation This tool works for matrices created in FlowJo, as the single stain controls are needed to calculate several statistics from to produce each value in the SSM. SSM’s can be calculated for a set of single stain controls in the Compensation Editor. It reflects the Poisson-based error in fluorescence quantification. The Spillover Spreading Matrix (SSM) is a measure of the performance of an instrument for a given panel.
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