In the last post, we looked at how to stabilize master mixes for flow cytometry panels. Today brings a quick follow-up to show an example of what doesn't work with this.
One of the big challenges in stabilizing the mix is working with the SIRIGEN polymers, particularly the Brilliant UV dyes. These interact with each other, and it's difficult to fully prevent these interactions even with the Brilliant Stain buffer. There are some ways to deal with this, including 1) titrating down the antibodies, 2) swapping conjugates around to find pairings that display reduced interactions and 3) using alternative dyes. The last option is really the easiest and most surefire way to do this, but there aren't currently alternatives for all conjugates.
Let's look at what happens if you don't do this. Here's a set of examples using antibodies without optimization and just storing the mix as suggested in the last post (dark tubes, tandem stabilizer, Brilliant Stain buffer). The PBMCs here are stained for an hour at room temperature with all the antibodies at the manufacturer's recommended test amount. If that mix is stored for two weeks, there are noticeable interactions between the BUV dyes (not surprisingly).
So, if you don't put in the legwork of testing the mixes for your panel, expect to run into problems.
From this, there's one other suggestion for how to limit these interactions: split the interacting partners into separate staining mixes. This can work if (as recommended), the expression on the cells for the BUV conjugate's target is low, reducing the amount of cell-bound BUV dye to create interaction sites for the second BUV dye. They're not in the same stored mix, so they won't interact while stored.
In the example below, the top row again shows what happens if we just chuck a bunch of antibodies in a vial and leave it for a couple weeks. On the bottom, those antibodies are pre-titrated and the interacting partners happen to be split into separate mixes because ICOS worked better overnight. There may still be a slight interaction, but it's definitely improved.
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