Why use spectral unmixing?
Spectral compensation utilise utilises signals from non-primary detectors. The additional information allows the software to separate individual colors colours more precisely, therefore improving resolutiondecreasing spillover.
The more light (data) you collect, the better the resolution will beunmixing can perform. It can be done on any cytometer as long as you collect n+1 data points where n is the number of colours in your cytometry panel.
Recording single stained controls:
You’ll record single stained controls just like conventional compensation. The same rules apply:
Positive controls have to be equally bright or brighter than the sample
All signals are within the linear detection range
The positive and negative populations must have the same auto-fluorescence (i.e. Pairing beads with beads with the same lot number, cells with cells)
Fluorochromes used for compensation must be the same as the sample (Can’t compensate AF647 with APC for example)
Additionally you you’ll want to:
Keep the parameters you are not using. Do not delete them!
Check if signals in their primary detectors are the brightest. If signals are brighter in non-primary detectors this may throw-off the matrix, resulting in artificial population spreadingwe have observed non-optimal spreading occurring.
Spectral compensation
...
in FlowJo
To use the spectral compensation feature, you you’ll need FlowJo version 10.6 .or later (FCS express now has this feature also)
Drag in the single stained controls.
Click the compensation icon and select the parameters you are using. Flow Jo should pick the positive & negative populations automatically. Adjust if needed.
...
Click on view matrix for calculation. You can then drag and drop the [M] icon to apply spectral compensation onto you sample.
...
Optimise weight weights may help reducing to reduce spread but it’ll take quite some time for calculation, depending on your computer compute power.
What if spectral compensation looks worse than traditional compensation?
This can be due to sub-optimal PMT voltage settings.
...