Intracellular Staining by Flow Cytometry
|Detection of Osteopontin/OPN in RAW 264.7 Mouse Cell Line by Flow Cytometry. RAW 264.7 mouse monocyte/macrophage cell line either (A) untreated or (B) treated with LPS was stained with Goat Anti-Mouse Osteopontin/OPN PE‑conjugated Antigen Affinity-purified Polyclonal Antibody (Catalog # IC808P, filled histogram) or isotype control antibody (Catalog # IC108P, 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.|
Osteopontin (OPN, previously also referred to as transformation-associated secreted phosphoprotein, bone sialoprotein I, 2ar, 2B7, early T lymphocyte activation 1 protein, minopotin, calcium oxalate crystal growth inhibitor protein), is a secreted, highly acidic, calcium-binding, RGD-containing, phosphorylated glycoprotein originally isolated from bone matrix (1). Subsequently, OPN has been found in kidney, placenta, blood vessels and various tumor tissues. Many cell types (including macrophages, osteoclasts, activated T cells, fibroblasts, epithelial cells, vascular smooth muscle cells, and natural killer cells) can express OPN in response to activation by cytokines, growth factors or inflammatory mediators. Elevated expression of OPN has also been associated with numerous pathobiological conditions such as atherosclerotic plaques, renal tubulointerstitial fibrosis, granuloma formations in tuberculosis and silicosis, neointimal formation associated with balloon catheterization, metastasizing tumors, and cerebral ischemia. Mouse OPN cDNA encodes a 294 amino acid (aa) residue precursor protein with a 16 aa residue predicted signal peptide that is cleaved to yield a 278 aa residue mature protein with an integrin binding sequence (RGD), and N- and O-glycosylation sites. OPN has been shown to bind to different cell types through RGD-mediated interaction with the integrins alpha v beta 1, alpha v beta 3, alpha v beta 5, and non-RGD-mediated interaction with CD44 and the integrins alpha 8 beta 1 or alpha 9 beta 1. Functionally, OPN is chemotactic for macrophages, smooth muscle cells, endothelial cells and glial cells. OPN has also been shown to inhibit nitric oxide production and cytotoxicity by activated macrophages. Human, mouse, rat, pig and bovine OPN share from approximately 40-80% amino acid sequence identity. Osteopontin is a substrate for proteolytic cleavage by thrombin, enterokinase, MMP-3 and MMP-7. The functions of OPN in a variety of cell types were shown to be modified as a result of proteolytic cleavage (2, 3).
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