Today's tip is something I learned from Anna Belkina at the Babraham Spectral Symposium. Let's look at how you can acquire multiple single-stained reference controls in SpectroFlo software on the Aurora. I had been doing this in a kludgy way using exporting and importing, and this is so much better.
First, why do we want multiple controls? The most common reason, I expect, is that you'll have a control that you aren't sure will work. This may be cells expressing a dim marker (e.g., ST2), a rare cell population (e.g., tetramer+ T cells) or a cell population that has a lot of autofluorescence (e.g., an eosinophil marker). In these cases, you'd do well to have a robust control like compensation beads or a different marker (e.g., CD4-PE instead of a PE tetramer). Personally, I prefer to have both types of controls--the ersatz and the real one matching my sample. This is particularly important for less stable fluorophores, such as tandems, where it may be impossible to perfectly replicate the signature of your sample with an alternative control. Another situation where you'd want multiple copies of each reference control is if you're testing options for the best unmixing, maybe comparing compensation beads. Finally, you could use this for directed autofluorescence extraction with slightly differing signatures between tissue sources (a topic for the future).
For this example, I'll run through the steps to set up three types of controls: cell controls (PBMCs), bead controls (using BioLegend's compensation beads) and Veri-Cells.
First, we create a Reference Group as per usual. I've set this to be my cells (PBMCs), with a universal negative set to my unstained, which is unstained PBMCs.
If I try to add more reference controls, I run into errors because they're duplicates of what I've already created. In this past, this is where I got stymied.
The trick is that each control for the same fluorophore needs to be unique in some way. This can be done by setting the "Control Type" options (e.g., beads vs. cells) or by changing the "Label".
For instance, I can add beads as a new Universal Negative and set my new controls to be beads under Control Type. Now I have no red flags.
Note that the third BV421 in the list is unlabeled, and that this is acceptable to the software because each of the other two is labeled and thus distinct. There's at least one factor differentiating each control.
Finally, I create another set for my Veri-Cell controls. If your controls come from different sources, it's really critical to match the Universal Negative correctly (or use an internal negative).
Hit save and proceed to acquisition.
When it comes to the unmixing, we now get options. Under "Control", we have a drop-down menu with the three controls that have been run for each fluorophore. I can select all bead-based controls:
Or I can mix and match controls, depending on which I find to be most accurate for each fluorophore (the example below is not intended to represent the best choice, just how to do this).
Hope that helps!