Flow Lab FAQs - no.2: Antibody Titration

Our second Flow lab FAQ is something we probably wish we were asked about more often... 

Antibody Titration -  How And Why?

Titration allows the identification of the optimal concentration of a given reagent in a particular experiment. It's worth doing because sometimes, the concentration recommended by the company, or in a paper, is not the best to use in your assay.

Why?

Titration will help you to find the best signal-to-noise ratio for your staining and therefore give you the best chance of detecting a weak signal. It may also save you money if the optimal concentration is less than the manufacturer's recommendation:

All fluorescence measurements have associated background noise, which comes mainly from two sources. Some intracellular molecules, like NADH, fluoresce when excited at certain wavelengths (autofluorescence). The flow cytometer also has inherent noise coming from detectors and electronics. 

The background noise can increase if the concentration of the dye/antibody is too high, because it will start binding not only to the high-affinity epitopes it's supposed to target, but also to low affinity ones (non-specific binding). In practical terms, the median of the negative signal will increase causing a drop in the staining index. You can see this drop in the green arrow on the plot below.

How:

Stain a known number of cells with a range of different antibody concentrations. For example, if the recommended concentration is 10μM you can test 2.5, 5, 10 and 20μM. Then analyse them on your flow cytometer. It is very important that the staining conditions are kept the same in the titration and in the experiment (incubation time, temperature, fixation, etc), because the staining level is affected by experimental conditions. The volume of the reaction is also critical, try to keep this constant.

 Which concentration should I choose after the titration?

 

To select which concentration (or range of concentrations) is the best to use, calculate the STAIN INDEX for each tested value. The stain index is calculated as follows:  

Use FlowJo or any other cytometry analysis software to calculate the median of the positive and negative signals and the standard deviation of the negative signal.

 

The concentration with highest stain index should be selected for use on the experimental panel. In the graph above, the best range of concentrations is marked with a red box.

Frequently Asked Questions

1.    Should I use a viability dye in the titration?

If possible, yes. Dead cells usually have higher levels of autofluorescnce and can bind antibodies non-specifically. 

2.    Can I use compensation beads for the titration?

No! With titration we are looking for the specific binding of an antibody to antigen. Compensation beads work by binding the light chains and the antibody NOT the specific binding site.

3.    My marker is really rare. Can I test the same fluorochrome targeting a more abundant marker?

No! We are trying to establish the antibody binding NOT the level of fluorescence. If the marker is rare in your cells, try to find a cell line where it is highly expressed; manufacturers datasheets will often say which cells have been used to test the antibody.

Remember - It is important to keep the same staining conditions for the titration and for the final experiment. 

Joana

2016 - A Big Year!

Not just because it has an extra day, but 2016 will be big for the Crick Flow Cytometry Facility. At some point in the middle of this year we will begin moving into the brand new Francis Crick Institute. Situated in the heart of London, next to St Pancras station and the British Library, the Crick will be one of one of the largest biomedical research Institutes in the world - you can read about that here.

For the past 18 months we have been preparing for the merger of the old London Research Institute and National Institute for Medical Research Flow Labs by aligning common practices and cross-training of staff and we are, we think, ready for the move. Because both Labs are large, we plan to move in phases so that we can keep operations running on both sites until all researchers make the move to the new building. It is logistically difficult but we like a challenge!

The Francis Crick Institute, London

In amongst all that, we need to make sure that we keep up with developments in the field - Twitter is a great source of information so via @citometria we were led to this article about using sound to sort cells. We will keep an eye (or maybe an ear) on that.

2015 was a good year for meetings - there was the first UK CYTO meeting for almost 30 years in Glasgow and then in November we had the biennial "Advances in Cytometry" meeting in London plus the usual mix of local flow meetings in London, Cambridge, Aberdeen, Nottingham and more. This year, we have CYTO in Seattle which is always a great place to meet up with old friends but also to see the new innovations in the field, I am always amazed at the breadth of cytometric applications and the passion with which people advocate the technology. But for those of you who can't make that, there is flowcytometryUK2016 which will be held in Leeds in July - planning is underway for that and there will be considerable Crick Flow Lab involvement - more on that later in the year.

We also hope to use this Blog to help promote cytometry education by putting up bite-sized answers to questions we often get from users, our Flow Lab FAQs - so bookmark this page or follow us on Twitter of course!

Derek