Getting more from titration

A quick tip today on antibody titration.

Titration helps us find the best signal to noise ratio for our antibodies, giving us better results in flow cytometry. If you're new to titration, I recommend reading this post, which contains a simple protocol, and these two great papers on the subject:

1. The Power of Reagent Titration in Flow Cytometry

2. Combinatorial antibody titrations for high-parameter flow cytometry

I think, though, that titrations can tell us more. In particular, a titration can tell you about how stable the panel is going to be if you use that antibody. For the CD40 in the first row below, the staining looks different at each concentration we pick. This means it will change easily in response to changes in cell number, staining time or staining temperature. It is not stable. The IgE staining, in contrast, looks very similar across a factor of ten. If you pick dilution factors where the staining looks the same at the next concentration up and the next one down, you build in a buffer that will give you much more stability between experiments and samples. This helps reduce batch effects.

Picking stable points in the titration curve tends to happen most of the time anyway based on the metrics we use. You can find a lot of examples of that in Diana Bonilla's paper. It is worth keeping in mind when selecting your concentrations, though, because there isn't always a stable plateau in an affordable region. The solution is either to proceed anyway with the knowledge that MFI values for the marker in question may be less reliable, or to put in the legwork of testing alternative combinations for your panel until you find one that only includes stable values.

Green-fronted Lancebill, Costa Rica