Designing a successful flow cytometry experiment requires effort on multiple fronts, including selecting the right antibodies for the experiment. The antibody selection process, often referred to as “building an antibody panel,” is the pre-experimental step researchers spend the most time on, and understandably so. If the cell sample to be studied is a low-abundance population or if the target under investigation is expressed in low amounts, for instance, designing an antibody panel that is not optimised for these factors can lead to experimental delays and wasted effort.
Herein, we discuss four questions every researcher should consider before building an antibody panel, as well as pointers for choosing between conjugated and unconjugated antibodies.
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4 Questions to Answer Before Designing Your Flow Cytometry Antibody Panel
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1. What is the goal of this experiment?
It is important to define clear goals for your flow cytometry experiment.
- Which cell populations are you studying during this experiment?
- Are there major subsets within these cell populations that you need to define to reach your cell of interest? In studying T cells, for example, you might select CD3+ as the epitope for the hematopoietic cell population and then differentiate them into CD8+ and CD4+ T cells.
- What is your prediction regarding the relative frequency of different cell types (low abundance versus high abundance populations)? Rule of thumb: low cell abundance and target expression often requires a brighter fluorochrome.
- Are you measuring a specific biological process/outcome (e.g. cell death, cell exhaustion, etc.)?
Being clear about the goal of your study helps with selecting the right antibodies/fluorochromes.
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2. Which fluorochromes can my instrument detect?
A plethora of flow cytometry analyzers exist on the market, from simple flow cytometers with a couple of lasers that can detect up to four colors, to instruments containing up to 10 lasers and which can detect over 50 colors. Understanding which colors your instrument can detect is also crucial to designing an effective antibody panel.
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3. What controls do I need to compensate for spectral overlaps and account for non-specific signals?
To collect accurate data for the cellular targets you’re studying, it is important to choose fluorochromes (colors) with minimal spectral overlap. Spectrally similar colors – like enhanced Green Fluorescent Protein (eGFP) and fluorescein isothiocyanate (FITC) – have overlapping emission spectra which can distort your data if, for example, you have a cell that expresses eGFP and choose to stain with a FITC-conjugated antibody. If you are using only two or three antibodies in your experiment, it is desirable to use colors far apart in wavelength to minimize overlap.
In addition, you need to ensure that what you are observing are on-target signals, not signals from non-specific binding of your antibodies. Therefore, it’s important to consider the controls for your experiment.
Figure 1: Spectral overlap and spectral distinction Commonly used controls for multicolor flow cytometry experiments include:
- An unstained control: Some cellular proteins emit natural fluorescence in response to being manipulated during the flow cytometry experiment. This can be picked up by a flow cytometer and provide a false signal. To check for and eliminate this “autofluorescence”, having an unstained control is useful.
- Single-stained controls: Cells or compensation control beads are stained with one antibody only to help calculate what the “true” signal for that antibody/fluorochrome might be.
- Fluorescence-minus-one (FMO) controls: These controls contain all but one of the staining antibodies and help define what it looks like when that fluorophore is missing from the staining mix.
- Isotype controls: An isotype control is an antibody that is of the same isotype and fluorochrome as your primary staining antibody. Using isotype controls can help researchers distinguish between specific and non-specific binding. Sometimes, antibodies can bind non-specifically to cell surfaces, to dead cells, or to Fc receptors present on the surface of cells. For the latter, researchers can address the problem by using an Fc blocking agent before staining with the antibody cocktail. The isotype control can therefore help with minimizing non-specific binding and interpreting target and non-target signals.
Without the controls appropriate for your experiment, you run the risk of misinterpreting flow cytometry data.
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4. What is my gating strategy?
A “gating strategy” is a sequential series of steps researchers use to isolate and study the cell populations they are interested in. Even for simple experiments, it is useful to exclude debris and cell doublets or clumps from the analysis. For complex cell populations, such as immune cell populations, gating becomes a critical component of data capture and analysis.
Therefore, to design an effective antibody panel, being clear on your gating strategy is crucial. Your gating strategy helps you define how you will resolve cell populations in your data analysis. A gating strategy example might look like:
- Identifying cells by size and granularity using forward/side scatter (FSC/SSC) to eliminate debris, cell doublets, and large aggregates.
- Performing compensation calculations based on your controls to distinguish signals from positive cells that were stained with the target antibody and negative cells that were not stained with the target antibody as well as resolve spectral spillover.
- Distinguishing between live and dead cells (and selecting live cells).
- Selecting a broad population marker with a population-specific antibody (e.g. CD3 for T cells or CD19 for B cells).
- Selecting and studying different population subsets within the general population (e.g., CD3+ T cells which are killer cells and express CD8, or helper T cells which typically express CD4).
Figure 2: Illustration of the idea behind a flow cytometry gating strategy for immune cells. After answering these questions, you have laid a solid foundation upon which to build your antibody panel. When it comes to selecting antibodies, researchers can choose between conjugated antibodies and unconjugated antibodies depending on their needs. Below, we discuss when it makes the most sense to choose one against the other.
Conjugated vs Unconjugated Flow Cytometry Antibodies
Conjugated flow cytometry antibodies are primary antibodies that have a fluorochrome, like fluorescein (FITC), phycoerythrin (PE), or allophycocyanin (APC), directly coupled to the antibody. Unconjugated antibodies on the other hand do not have a linked fluorochrome.
Figure 3: Illustration demonstrating the action of a conjugated primary (A) and an unconjugated primary antibody that requires a conjugated secondary antibody for detection (B).
Unconjugated Antibodies
If you choose to use unconjugated antibodies in your flow cytometry experiment, you will have to perform a secondary staining with an antibody that recognizes the primary and carries the fluorochrome, which increases the number of steps in your protocol compared to conjugated antibodies. However, unconjugated antibodies can be used across multiple types of experiments more easily than a conjugated antibody, adding flexibility. Additionally, a scientist who wants to conjugate their antibodies with specific fluorochromes that are not available commercially can do so with unconjugated antibodies, allowing for more customisation. Secondary staining is also known to amplify signals and can help with sensitivity if the target is expressed at low levels. On the other hand, they may also produce higher background signals.
Advantages of unconjugated antibodies for flow cytometry
- Higher signal sensitivity due to signal amplification.
- Increased flexibility since the same secondary can be used for different primary antibodies in various experiments.
- Commercial availability, as conjugated secondaries are more common than conjugated primary antibodies.
Conjugated Antibodies
Conjugated antibodies enable simple one-step staining of the cells you’re studying. This means using conjugated antibodies offer a simpler, faster workflow compared to unconjugated antibodies. They also lead to lower background signals compared to primary/secondary antibody combinations. They may cost less than having to purchase primary plus secondary antibodies. Conjugated antibodies are also useful for complex, multi-color panels allowing researchers to perform their experiments without cross-reactivity between secondary antibodies. In addition, if the sample you’re analysing is precious because perhaps the cell population is difficult to obtain, using conjugated antibodies in a shorter staining workflow helps you avoid multiple wash steps, and preserves the sample.
In summary, conjugated antibodies are preferred when the goal is rapid, reproducible, multiparameter flow cytometry with limited or valuable samples, whereas unconjugated antibodies are preferred when maximum sensitivity through signal amplification or greater experimental flexibility is required.
Advantages of conjugated antibodies for flow cytometry
- Faster, simpler, and possibly cheaper workflow without secondary antibody, reducing hands-on time.
- Reduced non-specific binding due to secondary antibody.
- Ideal for multicolor flow cytometry panels, eliminating cross-reactivity between secondary antibodies.
- Fewer wash steps, minimizing cell loss and preserving precious or limited samples.
- Reduced experimental variability, since the staining procedure involves fewer reagents and manipulations.
FluoSite™ Antibodies by BPS Bioscience
BPS Bioscience offers conjugated antibodies for your flow cytometry studies. Our FluoSite antibodies are tagged with fluorochromes using a method that is highly site-specific. The process ensures that the labeling site is far from the antigen-binding domain to preserve antibody functionality. The precise labeling strategy ensures the antibodies are useful for sensitive flow cytometry experiments. It also ensures homogeneity of the antibody preparation, lot-to-lot consistency, and experimental reproducibility.
Our FluoSite antibodies:
- Are recombinant and monoclonal
- Bind to human targets
- Have been validated for flow cytometry and
- Are purified from a human HEK293 expression system
Our long-term partner currently offers PE, FITC, and APC-labeled antibodies that bind to popular targets such as PSMA, KLK2, STEAP1, GPRC5D, CD19, CD20, CD4, CD8, EGFR, Claudin-18.2, and more, as well as isotype controls.
Figure 4: Cell surface expression of PSMA (prostate-specific membrane antigen) in PSMA CHO cell line (#79641) analysed by flow cytometry with FluoSite™ Anti-PSMA Antibody, PE-Labeled (#101976).
Looking for the right Flow Cytometry Antibody for your research?
Contact our team for help selecting the best solution for your research.
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References
Article content created by Tebubio using courtesy materials provided by BPS Bioscience.