Intracellular Staining by Flow Cytometry
|Detection of IL‑6 in J774A.1 Mouse Cell Line by Flow Cytometry. J774A.1 mouse reticulum cell sarcoma macrophage cell line treated with LPS was stained with Rat Anti-Mouse IL‑6 Fluorescein‑conjugated Monoclonal Antibody (Catalog # IC406F, filled histogram) or isotype control antibody (Catalog # IC005F, 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 central to the transition from acute inflammation to either acquired immunity or chronic inflammatory disease. It is secreted by multiple cell types as a 22 kDa-28 kDa phosphorylated and variably glycosylated molecule (1-4). Mature mouse IL-6 is 187 amino acids (aa) in length and shares 42% and 85% aa sequence identity with human and rat IL-6, respectively (5). Alternate splicing generates several isoforms with internal deletions (6). Mouse IL-6 is equally active on rat cells (7). 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 (8). gp130 is also a component of the receptors for CLC, CNTF, CT-1, IL-11, IL-27, LIF, and OSM (9). Soluble forms of IL-6 R are generated by both alternate splicing and proteolytic cleavage (9). 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, 10).