Antibodies 101: Beyond Surface Labeling

By Guest Blogger

Guest Blogger June 27, 2024

When it comes to labeling cells for flow cytometric analysis, the most common method is a cell surface label, where fluorophore-conjugated antibodies directly bind to epitopes of interest that are found in the extracellular space. The targeted epitopes can be motifs within transmembrane proteins, such as receptors, or post-translational modifications on those proteins, like glycosylation patterns. Surface labeling is particularly useful for classifying and sorting cells by identifying lineage markers, like many of the cluster of differentiation (CD) proteins found on immune cells.

However, there are more techniques and strategies one can employ beyond simply incubating cells with single-fluorophore-conjugated antibodies. These methods can include labeling markers with multiple proteins/antibodies, targeting markers found within the cytosol/nucleus, or visualizing DNA instead of proteins. Some stains can diffuse through the cell membrane freely, while others require a permeabilized membrane to enter cells. Depending on the research question and downstream application, one or several of those techniques can be used alongside standard surface labels. Here, I will introduce you to a few useful labeling and staining techniques beyond standard surface labeling.

Pro tip! Many people use 'labeling' and 'staining' interchangeably to refer to any technique that combines a marker with a signal. For clarity, Addgene uses 'labeling' to refer to antibodies and 'staining' to refer to small molecule dyes.

Indirect labeling

While antibodies used for target marker detection are usually conjugated to a fluorophore, not every marker will have a corresponding fluorophore-conjugated antibody commercially available. In other cases, fluorophore-conjugated antibodies may exist, but only with a limited selection of fluorophores. If the available colors are already reserved for other markers in your panel, and thus cannot be used, you may want to look at other methods. In the above cases, a good alternative is using a two-protein detection system, as is commonly used in western blotting or ELISAs.

A popular method for indirect labeling employs a primary epitope-targeting antibody that is conjugated to a biotin tag but not a fluorophore. In a separate step, cells are incubated with a fluorophore-conjugated streptavidin protein, which binds the primary antibody through the biotin-streptavidin interaction (Figure 1a). This interaction brings the fluorophore in proximity to the target marker, allowing for its detection. The advantage of this system is that streptavidin conjugates are commercially available for a wide range of fluorophores, allowing for great flexibility when it comes to fluorophore selection for panel design.

Find streptavidin plasmids and biotin plasmids at Addgene!

a) a ligand in a cell membrane, with an antibody bound to the extracellular side of the ligand. The antibody has a biotin tag on it, that is bound by a fluorescent molecule. b) the same antibody and ligand binding pair, but this time the antibody is bound by a secondary antibody with a fluorophore on it.

Figure 1: Indirect detection of target markers can be achieved through two-protein labeling by utilizing
(a) biotin-streptavidin binding and (b) antibody host species interactions. Created using biorender.com.

Another way of indirect labeling is through antibody host species reactivity. Just like for western blotting, a primary epitope-targeting antibody can originate from a range of species; for example, rabbit or mouse. In a second step, a fluorophore-conjugated secondary antibody is added that binds the conserved, host species-specific (“anti-rabbit”) region of the primary antibody (Figure 1b). As above, the advantage of this system is increased flexibility for panel design. Another perk is that one primary antibody can be bound by several secondary antibodies, increasing the fluorescence signal intensity. However, you need to be careful when selecting the primary antibody host species, as it has to be different from the other antibodies in your panel. Otherwise, your secondary antibody will bind to multiple primary antibodies, yielding a false positive signal.

Dump gating

When you are working with heterogeneous cell mixes, like bulk lympho-/splenocytes, you might only be interested in one of the various cell populations; for example, CD8+ T (“T killer”) cells. Typically, you would label the CD8 co-receptor protein on the surface of the CD8+ T cells and gate on the positive events for your flow analysis. However, certain downstream applications, like an in vivo adoptive transfer of those CD8+ T cells, require the cells to be minimally altered, so you would not be able to use antibodies that block surface proteins. In that case, you would want to look at negative selection: labeling markers that your cells of interest do not have, and selecting for the cells that are not labeled.

An easy and quick method of negatively selecting your cells of interest is magnetic bead-activated cell sorting, or MACS. But MACS usually only allows for sorting based on a single (lineage) marker, while fluorescence-activated cell sorting (FACS) enables you to include further (non-lineage) markers alongside, e.g., for cellular activation. In addition, FACS achieves greater purity of your sorted cell sample than MACS.

An effective strategy to sort out your cells of interest via FACS using negative selection is called dump gating. Let’s say we want to sort out all CD8+ T cells from a mix of CD8+ T cells, CD4+ T cells, B cells, and dendritic cells. In this case, we simply need to label the lineage markers of all cell types except CD8+ T cells. We might choose the markers CD4, CD19, and CD11c, which would label everything but the CD8+ T cells (Figure 2). Even better, we can use the same fluorophore for the three lineage markers we have chosen. As a result, all cells positively labeled with the selected color will not be CD8+ T cells and we can simply “dump” them by gating on the negative population.

Four different cells together. Three cells are each bound by an antibody with a red fluorescent probe. All three have arrows pointing to a trash can. The four cell is not bound by any antibody and has an arrow pointing to an open Eppendorf tube.

Figure 2: Dump gating labels all unwanted cell lineages with the same fluorophore to easily gate on the unlabeled cells of interest while the rest can be excluded, or “dumped”. From left to right: B cell, CD4+ T cell, dendritic cell, CD8+ T cell. Created using biorender.com.

Intracellular labeling

Oftentimes, a marker of interest is not located on the cell membrane but inside the cell. Due to their chemical nature, antibodies cannot penetrate the cell membrane; hence, intracellular labeling with antibodies requires chemically pre-treating the cells to allow for entry of the antibodies. Here, a two-step process is employed: the cells are first fixed and then permeabilized, using two different buffer solutions. Fixing causes protein cross-linking, killing the cell but preserving the cellular state (think of mummification). Permeabilization, as the name suggests, perforates the cell membrane to allow for antibodies to enter the cell (Figure 3). A useful side effect of the fixing/permeabilization (fix/perm) treatment is that the cells are now stable for much longer, even at room temperature, allowing for a flow analysis to be shifted to a later day. Note that intracellular labeling must be performed only after viability staining and surface labeling are already done.

Intracellular labeling can be subdivided into two categories, depending on the location of the targeted markers. Some commercially available fix/perm kits allow for labeling of cytosolic proteins, as well as proteins of the secretory pathway. Those can include cytokines and chemokines; enzymes like kinases, phosphatases, and ubiquitin ligases; and many more. This process, however, leaves the nucleus intact. If markers of interest are located inside the nucleus, a different fix/perm kit is required (to be precise, the fixing solution is different while the permeabilization solution can be the same). Examples of such targets include transcription factors, histones, and DNA repair enzymes. Note that when using the nuclear fix/perm buffer to label intranuclear proteins of interest, you can label cytosolic proteins alongside the intranuclear ones. You do not need to use a separate fix/perm cytosolic procedure in that case.

A cell with breaks in the cell membrane and nucleus membrane. Antibodies, attached to fluorescent dyes of different color, are entering the cell. There are two antibodies bound to proteins inside the cell, and one antibody bound to a protein inside the nucleus.

Figure 3: For intracellular labeling cells need to be fixed and permeabilized prior to incubation with the labeling antibodies. Only then can the antibodies bind to their targets in the cytosol and nucleus. Created using biorender.com.

Dye stains

Dye stains are non-antibody-based stains that bind DNA or free amines of proteins. Depending on the application, they can be membrane-permeant or membrane-impermeant, and the cells of interest can be the ones that are positive or negative for the dye. The main applications of dye stains in flow cytometry are cell proliferation and cell viability. Unlike intracellular labeling, dye staining can be done on live cells.

For cellular proliferation, esterified dyes that can enter cells freely are typically used. Inside the cell, the ester group is cleaved off by enzymes, turning the resulting molecule fluorescent and membrane-impermeant. With each cell division, the (invariant) amount of dye is reduced per cell (Figure 4a), yielding a stepwise decrease in fluorescence intensity in a (histogram) flow plot (Figure 4b). Through this method, differences in proliferation between cell populations can be visualized, achieved through, for example, a previously induced gene knockout. Besides amine- and DNA-binding dyes, nucleoside analogs like bromodeoxyuridine (BrdU) can be used. These dyes are incorporated into the DNA during DNA synthesis and then diluted with subsequent rounds of DNA synthesis (cell divisions).

a) a cell, with arrows pointing to two cells, which in turn have arrows pointing to four cells, representing cell replication. b) a graph with Count on the Y axis and Proliferation Dye on the X axis. The graph is a series of peaks that get higher as it moves from d4 (day 4) to d0 (day 0).

Figure 4: Proliferation dyes can visualize proliferative capacity and speed of cells. (a) With each division half of the dye molecules are lost and the fluorescence signal weakens. (b) The heterogenicity of proliferative behavior in a cell population can be seen as a stepwise decrease in signal strength in a flow plot. d = days post treatment. Created using biorender.com.

Cell viability dyes are membrane-impermeant and can only enter cells with compromised plasma membranes — i.e., dead cells — while live cells are protected. Amine-binding viability dyes can still bind free amines on the cell surface, which yields a much weaker signal on live cells compared to dead cells. As a result, the largely negative live cells can still be easily gated on during the analysis.

Conclusion

Congratulations! You made it through this blog post and hopefully learned a few new methods of target visualization beyond the simple surface labeling. Let’s briefly walk through the topics we covered in this post.

Besides direct labeling, indirect labeling can be used when suitable fluorophore-conjugated antibodies are not available for your marker of interest. Indirect labeling is achieved through protein-tag or protein-protein interactions, like biotin-streptavidin binding and antibody host species reactivity. When sorting cells with a high resolution for specific downstream applications, while maintaining a clean cell surface, dump gating is a useful way to get rid of undesired cell populations. Dump gating uses the same color for different markers, making it easy to gate on the negative cell population. To detect non-surface markers, a separate intracellular labeling protocol is necessary, requiring chemical treatment of the cells after surface labeling. Intracellular labeling can be performed on cytosolic and/or nuclear markers. Lastly, stains, based not on antibodies but on small molecule dyes, are useful, most notably when it comes to viability and proliferation staining. Dye stains are membrane-permeant or -impermeant and bind DNA or free amines, depending on the application.

Paul Heisig is a Research Associate in the lab of Arlene Sharpe at Harvard Medical School. His projects include investigating negative regulators of T cells and cytokine signaling in tumor immunity. When he's not in the lab, Paul enjoys weight lifting, sailing, and reading.