Intracellular Staining by Flow Cytometry
|Detection of IL‑6 in Human PBMCs by Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) treated with LPS were stained with Mouse Anti-Human IL‑6 PE‑conjugated Monoclonal Antibody (Catalog # IC206P, filled histogram) or isotype control antibody (Catalog # IC0041P, open histogram). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.|
Interleukin 6 (IL-6) is a pleiotropic alpha -helical cytokine that plays important roles in acute phase reactions, inflammation, hematopoiesis, bone metabolism, and cancer progression. IL-6 activity is essential for the transition from acute inflammation to either acquired immunity or chronic inflammatory disease. It is secreted by multiple cell types as a 22-28 kDa phosphorylated and variably glycosylated molecule (1-4). Mature human IL-6 is 183 amino acids (aa) in length and shares 41% aa sequence identity with mouse and rat IL-6 (5). Alternate splicing generates several isoforms with internal deletions, some of which exhibit antagonistic properties (6-9). Human IL-6 is equally active on mouse and rat cells (10). IL-6 induces signaling through a cell surface heterodimeric receptor complex composed of a ligand binding subunit (IL-6 R) and a signal transducing subunit (gp130). IL-6 binds to IL-6 R, triggering IL-6 R association with gp130 and gp130 dimerization (11). gp130 is also a component of the receptors for CLC, CNTF, CT-1, IL-11, IL-27, LIF, and OSM (12). Soluble forms of IL-6 R are generated by both alternate splicing and proteolytic cleavage (3). In a mechanism known as trans-signaling, complexes of soluble IL-6 and IL-6 R elicit responses from gp130-expressing cells that lack cell surface IL-6 R (3). Trans‑signaling enables a wider range of cell types to respond to IL-6, as the expression of gp130 is ubiquitous, while that of IL-6 R is predominantly restricted to hepatocytes, leukocytes, and lymphocytes (3). Soluble splice forms of gp130 block trans-signaling from IL-6/IL-6 R but not from other cytokines that utilize gp130 as a coreceptor (4, 13).
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