To provide measurability, most flow cytometry assays must also directly or indirectly have a label incorporated. Labels can include enzymes, fluorophores, or biotin. There are multiple methods to stain antibodies for flow cytometry. The choice of which method to use in your flow cytometry assay will depend on your cell type, antigens, and flow cytometry reagents.

Explore the flow cytometry application guide to enhance your research.

Direct vs indirect staining

In direct immunofluorescence staining, cells are incubated with an antibody directly conjugated to a label (eg FITC). Using directly conjugated antibodies requires only one antibody incubation step and eliminates the possibility of non-specific binding from a secondary antibody.

In indirect staining, the primary antibody is detected by a labeled secondary antibody. This second reagent may be an antibody with specificity for the first antibody. Alternatively, the avidin-biotin system can be used, where the primary antibody is conjugated to biotin and detected with fluorochrome-labeled avidin.

Key steps in both direct and indirect staining protocols include proper flow cytometry antibody selection, incubation, and washing to ensure reliable results and optimal detection of specific antigens on the cell surface. Validated target specificity of antibodies is essential for accurate and reliable flow cytometry results, as it ensures that the antibodies bind only to their intended antigens and provide trustworthy data for analysis of individual cells.

Figure 8. Direct and indirect labeling methods for flow cytometry

Benefits and limitations of direct and indirect antigen detection

Both direct and indirect methods for antigen detection each have benefits and limitations (Table 1). The direct method is typically preferred for flow cytometry experiments to save time and for ease of use. These factors are also relevant for multicolor experiments; however, using secondary antibodies can provide signal amplification¹.

Table 1. Comparison of direct and indirect antigen detection methods.

Intracellular staining

Protocols for the staining of intracellular antigens for flow cytometry utilize various fixation and permeabilization methods to allow antibodies access to internal cellular proteins. Detection of these intracellular proteins is typically achieved using fluorescently labeled antibodies and a range of fluorescent dyes, which provide flexibility in experimental design and multiplexing. Flow cytometry can be used to analyze not only cells but also other particles such as vesicles, enabling the study of diverse biological entities. Successful staining depends on optimizing experimental conditions through titering of antibodies, appropriate controls to set up the flow cytometer correctly, and optimized fixation and permeabilization procedures².

Detection of secreted proteins

The detection of secreted proteins is difficult as the protein will be released from the cell before detection or may degrade rapidly. Drugs that interfere with secretion, such as Brefeldin A, can be used to inhibit the secretion of expressed proteins by retaining them in the Golgi apparatus. The intracellular staining method can then be used to detect the target protein.

Understanding fluorophores

Fluorophores, also known as fluorochromes, are critical for detection in flow cytometry. A wide range of fluorescent dyes is available for flow cytometry, each with distinct spectral properties that allow for multiplexing and flexibility in experimental design. Understanding their properties is essential for correcting for fluorescence spillover in multicolor experiments. Fluorophores are commonly used with antibodies as fluorescence detection reagents in flow cytometry. They absorb and emit light within a range of wavelengths, generally referred to as the absorbance (excitation) and emission spectra (Figure 9)³.

Figure 9. Fluorophore excitation and emission

Tandem dyes for flow cytometry

The design of multicolor panels often requires tandem dyes consisting of two different, covalently attached fluorophores. One fluorophore (donor) is excited at a particular laser wavelength, while the other emits light at a different wavelength. For example, in phycoerythrin-Cy7 (PE-Cy7), PE and Cy7 act as the donor and acceptor fluorophores, respectively. Therefore, PE-Cy7 will have the excitation characteristics of PE and the emission characteristics of Cy7 (Figure 10).⁴

Figure 10. Tandem dye excitation and emission.

Tandem dyes increase your panel size and flexibility because several fluorophores can be excited by a single laser and measured by different detectors. For example, Alexa Fluor® 488, PE, PerCP-Cy5.5, and PE-Cy7 are excitable with a blue laser (488 nm). However, they will produce green, yellow, purple, and infrared emissions (Figure 11).

Figure 11. Excitation and emission spectra for a range of tandem dyes.

Hints and tips for handling tandem dyes

• Always protect tandem dyes from light as they are highly susceptible to photobleaching

• Do not freeze tandem dye antibody conjugates as this may result in denaturation of the donor fluorophore

• Minimize sample fixation or permeabilization as much as possible as this reduces their brightness

• Each batch requires optimization due to high batch-to-batch variation

• Some bleed-through emission from the donor might be observed since FRET efficiency is never 100%

• Label cells at 4°C to avoid degradation or decoupling of tandem dyes

Selecting a fluorophore conjugate for flow cytometry

The antibody’s ability to resolve a positive from a negative signal in flow cytometry often depends on the fluorophore used. It is important to review the available options for secondary antibodies and fluorophore conjugates, considering factors such as species, subclass, isotype, and label. When planning your experiment, be sure to select the appropriate fluorophore conjugate based on your specific experimental requirements to ensure optimal results.

Table 2 is a general guide to the properties of various common fluorophores. Further to this, there are some differences in the relative intensities of each fluorophore, depending on the specific flow cytometry antibodies used.

Table 2. Properties of common fluorophores used in flow cytometry

Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

References

1. Serke, S.,, Lessen, A.V.,, Huhn, D. Quantitative fluorescence flow cytometry: a comparison of the three techniques for direct and indirect immunofluorescence Cytometry 33 (2),179-87 (1998)
2. Mahnke, Y.D.,, Roederer, M. Optimizing a multicolor immunophenotyping assay Clinics in Laboratory Medicine 27 (3),469-485 (2007)
3. Horvath, G.,, Petras, M.,, Szentesi, G.,, et al. Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements Cytometry A 65 (2),148-157 (2005)
4. Le Roy, C.,, Varin Blank, N.,, Ajchenbaum Cymbalista, F.,, et al. Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism Cytometry A 75 (10),882-90 (2009)