Cytometry and Antibody Technology

Combinatorial Antibody Titrations

by | Feb 27, 2024 | Uncategorized | 0 comments

Greetings from our absolutely delightful and unseasonably warm day for February – I hope you are getting, or did get, a chance to be outside. Our temperature is going to drop 40 ° F by tomorrow….Such is weather in the Midwest.

This post will cover a recent paper in Flow Cytometry A about antibody titrations. Dr. Burn, Dr. Mair, and Dr. Ferrer-Font will quickly become the fav PI’s for grad students the world over when they learn how to spend less time in front of the cytometer on customization.

A brief word about titrations before we start. I approximate the importance of Ab titrations to that of annealing temps for primers – In both cases, the wrong values will make them either stick to everything, or nothing at all. It is an example in research where you want to be as specific as possible to ensure good results. A correct Ab titer will allow for the best separation between positive and negative populations while minimizing the spread of the negative population and reducing non-specific binding – in the world of Flow cytometry, it is as important as adhering to all the other rules for panel design.

So given its importance, why don’t we do it more regularly? Time probably. On a spectral panel with 25 stains, this is hours of work of work setting up and analyzing 150 titration data points; OR you could ask your lab bench mate ‘Hey, what do you use for CD45?’; OR you could see what the article you’re trying to recapitulate used; OR you can just wing it…. I’ve been in research long enough to be able to share with you (not proudly of course) that I have done all of those things.

What if there was a way to expedite this process? As it happens, non-overlapping markers can be titered and data collected by doing them in groups of four or five. How is this possible? Well, remember that the stain index is calculated as (SI=(Median Fluorescence pos-Median Fluorescence neg)/(rSD negative x2)), which means that if you are distributing your Abs over the whole spectrum of the instrument (let’s assume a 5L Aurora), having one on the BUV, one on the Violet, Blue, etc., then you could reliably look at 4 or 5 at a time, and calculate the stain index accordingly. The caveat here is picking non-overlapping fluorophores. BV711 and AF700 might not be the best to run together, but BUV 395, AF 532, BV 421 and something off the YG and R laser of your choosing ought to work fine. While there would still be work to be done in combining, say, a 25 marker panel, by breaking the titrations into groups, you can have a very manageable 50 sample analysis versus 150 sample analysis. Outside of being mindful of the presumed expression level of your markers, it ought to be straight forward.

Finally – a word on titration templates. Both Flowjo and FCS express have plugins to calculate your SI values. Conversely, you can do them all yourself in an Excel doc which is what I still do since that’s how I learned to do it – but I would give FCS express and Flowjo a look for this process – I’ll add links to the respective youtube tutorials.


Burn OK, Mair F, Ferrer-Font L. Combinatorial antibody titrations for high-parameter flow cytometry. Cytometry A. 2024 Feb 6. doi: 10.1002/cyto.a.24828.

FCS Express Ab titration:

FlowJo Ab titration:


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