|IL‑6 in Human PBMCs. IL‑6 was detected in immersion fixed human peripheral blood mononuclear cells (PBMCs) stimulated with LPS and monensin using Mouse Anti-Human IL‑6 Monoclonal Antibody (Catalog # MAB2061) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (yellow; Catalog # NL007) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.|
|Cell Proliferation Induced by IL‑6 and Neutralization by Human IL‑6 Antibody. Recombinant Human IL‑6 (Catalog # 206-IL) stimulates proliferation in the T1184.108.40.206 mouse plasmacytoma cell line in a dose-dependent manner (orange line). Proliferation elicited by Recombinant Human IL‑6 (2.5 ng/mL) is neutralized (green line) by increasing concentrations of Mouse Anti-Human IL‑6 Monoclonal Antibody (Catalog # MAB2061). The ND50 is typically 0.05-0.15 µg/mL.|
Intracellular Staining by Flow Cytometry
Detection of IL‑6 in Human PBMCs by Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) treated with |
100 μg/mL LPS for 24 hours were stained with Mouse Anti-Human IL‑6 Monoclonal Antibody (Catalog # MAB2061, filled histogram) or isotype control antibody (Catalog # MAB004, open histogram), followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0102B). 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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