Intracellular Staining by Flow Cytometry
|Detection of IL‑17F in Mouse Splenocytes by Flow Cytometry. Mouse splenocytes were treated for 24 hr with 50 ng/mL PMA, 200 ng/mL Ca 2+ ionomycin, 10 ng/mL Recombinant Human TGF‑ beta (Catalog # 240‑B), and 40 ng/mL Recombinant Mouse IL‑6 (Catalog # 406‑ML), then stained with Rat Anti-Mouse IL‑17F Monoclonal Antibody (Catalog # MAB2057, filled histogram) or isotype control antibody (Catalog # MAB006, open histogram), followed by Phycoerythrin‑conjugated Anti-Rat IgG F(ab')2 Secondary Antibody (Catalog # F0105B). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.|
The Interleukin 17 (IL-17) family proteins, comprised of six members (IL-17, IL-17B through IL-17F), are secreted, structurally related proteins that share a conserved cysteine-knot fold near the C-terminus, but have considerable sequence divergence at the N-terminus. With the exception of IL-17B, which exists as a non-covalently linked dimer, all IL-17 family members are disulfide-linked dimers. IL-17 family proteins are pro-inflammatory cytokines that induce local cytokine production and are involved in the regulation of immune functions (1, 2).
Mouse IL-17F cDNA encodes a 153 amino acid (aa) protein with a putative 20 aa signal peptide. Among IL-17 family members, IL-17F is most closely related to IL-17 sharing approximately 46% aa sequence identity. Mouse and human IL-17F share 55% sequence identity. IL-17F is expressed in activated CD4+ T cells and activated monocytes. Two receptors (IL-17 R, and IL-17B R), which are activated by IL-17 family members have been identified. In addition, at least three additional type I transmembrane receptors with homology to IL-17 R, including IL-17 RL (IL-17 RC), IL-17 RD, and IL-17 RE, have also been reported (1, 2, 5). The functions for IL‑17 RC, D, and E are not known. Purified IL-17 R and IL-17B R do not bind IL-17F with high-affinity in vitro. However, binding of IL-17F is detected in cells transfected with IL-17 R, raising the possibility that a co-receptor may be required for IL-17F signaling through IL-17 R (4). The biological activities mediated by IL-17F are similar to those of IL-17. IL-17F stimulates production of IL-6, IL-8, G-CSF, and regulates cartilage matrix turnover by increasing matrix release and inhibiting new matrix synthesis (4). IL-17F also inhibits angiogenesis and induces production of IL-2, TGF-beta, and monocyte chemoattractant protein-1 in endothelial cells (3).
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