Antibodies for Flow Cytometry

Introduction to Flow Cytometry Antibodies

Flow cytometry antibodies are used to label specific proteins on or within cells to enable detection and quantification. These antibodies are typically conjugated to fluorescent dyes, allowing them to be excited by a laser and emit light detected by the instrument. The fluorescence intensity is then used to determine cell characteristics, such as their size, shape, and composition.

Fig. 1 Comprehensive immune profiling by multicolor flow cytometry panels. (Holmberg-Thyden S,et al., 2021)

Functions of Antibodies

In order to analyze specific targets by flow cytometry, fluorescent labeling of cellular components is required, either using individual fluorescent molecules (e.g., cytochromes) or fluorophore-labeled antibodies (either directly conjugated to antibodies or using conjugated secondary antibodies).

Antibody

Antibodies can specifically bind individual proteins or protein modifications and serve as a means to label those targets for detection. By labeling a target of interest, researchers can assess protein abundance or activity in various cell types, disease states, treatment conditions, developmental stages, or other biological models. Antibodies that bind to different proteins do not interfere with each other, so multiple antibodies can be used to detect multiple targets simultaneously. This is called multiple analyses. A limitation to the number of antibodies is the ability to assay each antibody independently of the other. In flow cytometry, this differential measurement is accomplished using fluorescent molecules called fluorophores. Successful experiments require highly specific and validated antibodies and reliable fluorophores.

Fluorophore

Protein fluorophores such as GFP are very popular because they can be encoded by DNA. By adding the DNA sequence of GFP to the DNA sequence encoding a specific protein, cells can produce the protein with its own fluorescent tag, allowing researchers to directly analyze the location and quantity of the protein in in vitro and in vivo systems without subsequent labeling operations. Fluorescent proteins such as GFP can also be selectively expressed in specific cell types or in response to specific conditional stimuli. The ability to make certain cells "self-report" using fluorescent markers provides the opportunity to selectively enumerate and/or sort these cells.

Direct and indirect fluorophore labeling

The fluorophore used to obtain the readout in a flow cytometry experiment is covalently attached to the antibody. This process is called conjugation, and this antibody-fluorophore pair is referred to as a conjugate. When using the conjugated antibody in a cellular assay, the light emitted by the fluorophore serves as a direct indicator of the amount of antibody present in or on the cell. By utilizing conjugates that specifically bind proteins or protein modifications, researchers can directly quantify the target of interest in each cell based on the level of fluorescence emitted by that cell. This process is called direct detection or direct flow cytometry.

Indirect flow cytometry involves the use of secondary antibodies directed against immunoglobulins in the host from which the primary antibody was generated. In this method, the secondary antibody is conjugated with a fluorophore. This method may be preferred over direct flow cytometry when primary antibody conjugation may result in spatial variations affecting specificity and function. This method can also be preferred when an increased signal is desired. Multiple secondary antibodies can bind to a single primary antibody, amplifying the signal derived from each antigen molecule bound by the primary antibody. However, indirect flow cytometry has a limited number of antibodies combined in one experiment. Each primary antibody must be incubated in a different species (such as rabbit, goat, or mouse) for the secondary antibody to have specific detection capabilities.

Direct flow cytometry is often preferred because it eliminates the extra steps required when secondary antibodies are required. It uses many different antibodies simultaneously, maximizes the amount of information collected, and provides greater insight into the cell population present.