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To calculate the compensation matrix from a series of single colour fluorescence controls, load all of the compensation controls into the playlist. Now, ideally, this would be in fluorescence detector order. It’s not absolutely essential to do it at this stage, but it does make life a little easier later on. Go to the compensation tab of the ribbon and select the compensation wizard. In the compensation wizard, select the parameters you wish to calculate the compensation for and deselect any parameters that you do not want to compensate for.
If you wish to gate on a plot, select which parameters you wish to gate on. This would usually be forward inside scatter. As in this case, we do not have a background fluorescence sample so we select this option. If you have a separate negative control tube, you would select the use negative control file option. You can also use the negative regions with a positively labelled sample. So, if you have positive and negative events in the same parameter, you can set and choose which one is which. In this case, we will be choosing the no background fluorescence option.
Moving on, you can choose histograms or a dual parameter density plot. If you have complicated overlapping compensation then sometimes it is easier to actually select the two parameter density plot. Usually, though, compensation can be done using histograms.
The next step is to assign a fluorescence parameter to each file. This is where putting your samples into the playlist in fluorescence order makes life a lot easier because now I don’t need to actually mess around changing any of these because they’re already correct. If they are incorrect, there is a drop-down where you can select which parameter goes with which file. Select next to begin the compensation process.
So we start with the first file. We move the gate around the population that you are interested in. This gate position will be used for all subsequent files so it shouldn’t need to be changed. And then, on the histogram, move the region over the compensation peak. If you are using a separate negative control file, you will first have to set the region over the negative peak for each parameter in the negative file. If you’re using positive and negative in the same file, you will have two regions to set: one around the positive and one around the negative for each file. In this case, we are using positively labelled samples, so we only have one region to set around the positively labeled events.
As we set the region, the compensation is calculated for each parameter and you can see those values in the table here and you can immediately see if there’s going to be any issues with the compensation. Press next to go to the second sample, make sure that the gate and positive event region are in the correct position before moving on to the next sample.
As we go through each sample, the table builds up for each parameter. Once complete, you can review the entire matrix, and you can print the matrix and save the matrix to file. At this point, you can apply the compensation file to the current playlist so it will apply these compensation settings to all files in the playlist, and this will enable you to check the calculated compensation values against the compensation controls.
The easiest way of checking is to use the previews and to do this, we need to set a gate on the forward scatter side scatter plot to pick up the single lymphocytes. And then we can use the previews just to quickly step through each sample and check that it looks properly compensated. If it turns out that there is a bit of a compensation issue, then you can adjust the settings using the manual methods we previously discussed. To verify compensation, load a sample that has been stained with all your required antibodies, and then set the compensation to be the compensation that has been calculated in the wizard.
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