|Figure 1. The use of the isotype control alone (yellow) may lead to an overestimation of the number of cells staining positive for intracellular IL-1 beta. The different staining patterns between permeabilized (red) and non-permeabilized (blue) cells represents intracellular cytokine staining.
Method: Peripheral blood (PMN) cells were stained for IL-1 beta after culture with 0.5 µg/mL LPS and 3 mM monensin. Cells were fixed and then permeabilized (or not), prior to staining with fluorescein-labeled anti-IL-1 beta (Catalog # IC201F).
|Figure 2. Surface-blocked, non-permeabilized cells (blue) have an antibody reactivity comparable to an isotype control, because they show only non-specific staining. The blocked, permeabilized cells (red) are clearly resolved as intracellularly stained cells. Comparison of the staining of surface blocked (Figure 2) vs. non-blocked (Figure 1) permeabilized cells clearly shows the contribution of surface antigen to the total antibody binding.
Method: Stimulated cells (see Figure 1) were first incubated with a non-fluorescent antibody (same clone as the staining antibody) prior to fixation, permeabilized (or not) and then stained with the fluorescent anti-IL-1 beta antibody.
The Th1 and Th2 paradigm of immune regulation1,2 requires a method for identification of such cells within a mixed cell population. Despite suggestions of Th1- or Th2-specific surface markers, most researchers rely on the production of Th1 or Th2 cytokines (e.g., Th1 cells produce IL-2 and IFN-gamma, while Th2 cells produce IL-4, IL-5, and IL-10).2 The frequency of cells that produce a particular cytokine can be measured by intracellular staining with labeled antibodies and flow cytometric analysis. A technical problem associated with this technique is the potential detection of surface-bound as well as intracellular cytokines.
When cytokines are secreted, they may bind to cell-surface receptors on either the secreting cell or other cells. This illustrates the need to distinguish between extracellular and intracellular cytokine staining. The use of isotype controls will simply reveal nonspecific staining. Controls are necessary for demonstrating the specific location of cytokines.
Staining of non-permeabilized cells controls for cell surface-bound cytokines. In Figure 1, stimulated PMN cells were stained with an IL-1 beta antibody in the presence or absence of a permeabilization reagent. Staining to the right of the isotype control represents specific positive cytokine staining. Staining of non-permeabilized cells may be due to cell surface-bound IL-1 beta. In contrast, the increased staining observed following cell permeabilization is due to the presence of intracellular IL-1 beta.
Staining of surface-blocked, permeabilized cells controls for intracellular cytokines. In Figure 2, pretreatment with an unlabeled blocking antibody was used to prevent cell surface reactivity. Staining of non-permeabilized cells after pretreatment is similar to staining with an isotype control, since only non-specific binding is revealed. Pretreated, permeabilized cells demonstrate specific staining of intracellular IL-1 beta.
The ability to detect both intracellular and cell surface-bound cytokines varies among antibodies. R&D Systems selects clones with reduced binding capacities to cell surface-bound cytokines. It is not possible, in all cases, to eliminate surface staining. We encourage researchers to select controls that aid in confirming intracellular cytokine expression.