Panel Design: Build a backbone

I suggest starting by designing what I call a backbone for your panel. This is just the basic gating markers that will allow you to identify the cell types you are interested in.

This backbone is useful for titrating and testing antibodies as well as determining where you will have good resolution for your activation markers.

An additional advantage of designing the backbone first is that these markers tend to be the most widely available, so you can design these with the newest fluorophores if you wish. New fluorophores tend to have limited options, so it’s easier to assign them first and later fill in slots with conjugates in more common colors.

Example of a panel "backbone":

This set of markers forms the basis of the panel, allowing us to identify the major cell types.

I recommend taking your backbone-stained cells and unmixing it to the full combination you intend to use. With this ten-color-stained backbone, unmixing to thirty colors looks fine. There is minimal loss of resolution at this point. This is like a twenty-color FMO; staining for ten colors with twenty empty channels.

Use library reference controls to test which fluorophores can be added without affecting resolution.

How To:

Use the library controls to rapidly and easily assess fluorophore combinations to use in the panel. First, get samples of any fluorophore you plan to use. Borrow, request free samples, order a trial size, whatever. The marker is unimportant at this stage. Store those as library/reference controls in the software (see instructions for Aurora here). Run your stained backbone sample(s) on the cytometer using appropriate single color controls for the markers present in the backbone (e.g., 10 colors in the above example). Edit the experiment, adding in various other fluorophores you think you'd like to use. Unmix these extra fluorophores using the stored controls, ticking the box. This way you don't have to run all the colors every time you do this. At this stage, using perfect controls for these empty channels in your panel isn't important because you don't actually have any staining there. As such, we care primarily about how well the negative resolves as opposed to the spread/spillover of the positive.

This unmixing approach allows us to rapidly determine how large we might wish to make the panel. By unmixing to 46 colors, we see that the resolution becomes impaired in certain areas, and this is still without adding in staining in those channels. After a certain extent, adding more markers will make the panel worse, so be sure each marker in your panel has a purpose.

We can take this backbone unmixed to 30 or 40 colours and look at all the NxN biplots to see where the design is causing spread, and where we have lots of clean space to work with.

It can be very helpful to use the backbone when doing antibody titration and testing as discussed here.