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

Flow Lab FAQs - no.1: Tandems

We've set ourself a new task for our monthly lab meetings - take one of the questions we're often asked by our users and answer it in less than five minutes, with no slides. Although most of it is stuff that we all know already, it's always good to have a refresher and it provides an opertunity to discuss how we explain things to users to make sure they get acurate and consistent information.

We'll be sharing some of these Flow Lab FAQs on this blog, first up:


What are Tandem Conjugates and what are their Pros and cons?

What are they?

Tandems are essentially two fluorochromes chemically coupled together:

 

With single fluorochromes we use a laser to excite the molecule to a higher excitation state, then look at the energy given off as the molecule returns to its ground state.

Tandem conjugates use FRET (Förster, or fluorescence, Energy Resonance Transfer) so that the energy emitted by the first fluorochrome (A, the donor dye) is passed to the second (B, the acceptor dye). B is then excited to a higher state before dropping back down and emitting energy that we can detect.

The Benefits:

The difference between the excitation wavelength and the emission wavelength of a fluorochrome is known as the Stokes Shift. The size of this shift is limited in single fluorochromes but can be greatly increased by using a tandem. This means that with limited excitation sources (lasers) you can make use of more of the visual spectrum.

The Problems

No matter how efficient the process of making a tandem, there will always be some residual fluorescence from the donor dye. If a sample is stained with PE and PE-Cy7 antibodies it will be impossible to tell the difference between that and the PE signal from the tandem.  Even worse, this changes over time. Fortunately we can do something about this as we know it will happen. We also need to take more care with the storage of tandem dyes as their emission spectra will change over time.

Precautions To Take When Working with Tandem Conjugates

Controls

Controls

Controls

·      When using a tandem, ALWAYS have a single colour control

·      Always use the exact same antibody in your control as in your panel.

o   Different batches of tandems will always be slightly different so it is essential for correct      compensation that the exact same batch is used for the controls and the sample.

o   If your population is too rare or expressed at too low a level for a good control, use beads instead of cells

·      Always check the single control in the channels for its component parts to see donor emission.

·      Tandems tend to be more photosensitive than single fluorochromes so need to be protected from light during storage and during and after staining.

·      Tandems are also more prone to fixation issues as fixation will always alter the chemical structure of the dyes so make sure the controls and samples are treated in the same way.

·      When you get a new batch of a tandem, it’s a good idea to run it and look at the spillover, e.g. PE-Cy7 spillover in the PE channel. Then follow this over time. If the spillover starts to increase the efficiency of the FRET in the tandem is probably becoming less.

·      The name of most tandems makes it obvious what they are but that isn’t the case for some newer fluorochromes. Eg. Brilliant Violet BV 605 is a tandem of BV421 and Cy3.5 (all BV dyes above 605 are tandems). All the same precautions should be taken with these less obvious tandems.

Kirsty