Antibody Titration in Flow Cytometry

Gain Confidence to Design
Flow Cytometry Experiments

To Boldly Flow - Have confidence to start flow cytometry

To Boldly Flow - Have confidence to start flow cytometry

 Download PDF

Flow cytometry is a powerful technique widely used in biological research, but if you are just beginning your flow journey, it can be hard to know where to start.

To make this easier, we have created a step-by-step guide to starting flow cytometry with useful information and tips to help you plan, execute and interpret your experiments, ensuring you get the best results.

We also demonstrate how the panel builder tool can help you design your experiments, no matter how many colors you plan to use.

Select the steps of interest:


Know Your Instrument Know Your Instrument
 

If you don’t know which lasers and filters are available, you are likely to get data you can’t analyze. Our panel builder contains all of the laser and filter settings of common instruments, as well as being fully customizable, enabling you to choose the right fluorophore for your instrument.

Related instruments:

Related pages:

Multicolor panel builder

Principles of the flow cytometer

Back to top

Sample Preparation Sample Preparation
 

As with any experiment, decide on the sample you want to analyze, then how you are going to create the single cell suspension critical for flow. Sample preparation is key to success as poor samples will only give poor results. Simple things like whether the sample is frozen or fresh, adherent or in suspension and whether anticoagulants and removal of red cells are needed or not, should be considered. Looking after your cells post-harvest is also crucial; consider the cell concentration and storage temperature to keep your cells healthy.

Back to top

Marker Expression Marker Expression
 

Identification of cells through their marker expression is one of the key techniques in flow cytometry. Some cells are easy to identify using one or two markers, however some subsets require identification of multiple markers to accurately identify a cell subset. Moreover, if you want to identify memory status, activation state and cytokine profiles, the number of markers can significantly increase. To help you find the right marker, we have a marker selection tool, with maturation pathways for both human and mouse.

Another important consideration is the level of marker expression or antigen density. This will influence your choice of fluorophore as dim fluorophores should be paired with highly expressed markers and vice versa. Use the antigen density function of the panel builder to help choose the best fluorophore for your experiment.

Related pages

Back to top

Cell Frequency Cell Frequency
 

Cell frequency can influence both the choice of fluorophore and how many cells you need to collect to obtain meaningful data. If you are searching for rare cells such as stem cells, or have a complex gating strategy, you may have to collect many more cells compared to more common cell types such as T cells.

Related pages

Cell frequencies of common samples

Back to top

Choosing a Fluorophore Choosing a Fluorophore

Choosing a fluorophore rapidly becomes complicated as you increase the number of fluorophores. As already noted, fluorophore excitation and brightness depends on your cytometer and your cell of interest’s abundance. However, choosing a fluorophore can depend on many other factors, including emission wavelengths as overlapping emission spectra affect staining and require compensation. The recommended way to avoid compensation and obtain the best resolution is to separate out fluorophores as much as possible across lasers and filters. With pre-loaded cytometer settings and the ability to exclude incompatible fluorophores, our panel builder can help you choose the best options.

Back to top

Choosing the Right Antibody Choosing the Right Antibody
 

Using antibodies conjugated to fluorophores, cells from virtually any source can be identified. However, finding the right antibody can be challenging. Search our website to find antibodies by marker, clone, isotype and target species. Alternatively use our panel builder to find antibodies with the right fluorophore. Once you have sourced your antibody, and before starting your experiment, we recommend titration of the antibody. This can improve your data, by reducing the levels of background staining whilst maintaining a bright, positive population, and save you money! If you can’t find the antibody conjugated to the fluorophore you want, you could use a secondary antibody or a conjugation kit depending on availability and complexity of staining.

Back to top

Controls Controls
 

Controls are essential in any experiment to confirm positive results from background. In flow cytometry these can be biological, positive, negative, viability, isotypes, Fc blocking and fluorescence minus one controls depending upon your experiment. Dead cells bind antibodies non-specifically therefore it is essential to remove them from your analysis. The use of forward and side scatter may not always be sufficient. Viability dyes are available in a wide range of excitation and emission wavelengths; easily fit controls to your experiment using our multicolor panel builder.

Back to top

Staining Protocol Staining Protocol
 

Depending on the location of your antigen on your cell, the staining protocol may change. Surface staining with antibodies can be relatively straightforward but intracellular staining requires fixation and permeabilization, and a golgi inhibitor for cytokine staining. There are various methods and reagents for fixation and permeabilization. Depending on the antigen or technique being performed, these may also require optimization. Use the panel builder to create dump channels to improve resolution by excluding unwanted cells.

Back to top

Data Analysis Data Analysis
 

Once you have collected your data, don’t be afraid to play around with it and try different gates during analysis. Remember to remove dead cells using a viability dye rather than forward and side scatter. Removing doublets is also an essential part of your analysis to avoid false positives, particularly when looking at the cell cycle.

Back to top

Flow cytometry does not have to be difficult, and considering these critical steps before you start will improve your chances of obtaining great results. As mentioned, the multicolor panel builder is not only a useful tool to build large panels but also to:

  • Understand your cytometer
  • Choose and separate out fluorophores by lasers and detectors
  • Include compatible viability dyes
  • Select flow validated antibodies
  • Exclude unwanted cells using a dump channel

Watch the video tutorial and try the tool today and see how it can help you.

Bio-Rad has over 4,000 flow cytometry validated antibodies and flow cytometry resources to help you with your experiments. These include panel building for myeloid and B cell subset identification, as well as flow cytometry for techniques other than immunophenotyping, such as apoptosis and proliferation. For helpful tips and key information on flow techniques or more guidance on flow cytometry, take a look at our resources and reagents below.

Related flow cytometry products


Resources


Back to top