Intracellular Staining by Flow Cytometry
|Detection of IL‑17D in Mouse Splenocytes by Flow Cytometry. Mouse splenocytes were stained with Rat Anti-Mouse IL‑17D PE‑conjugated Monoclonal Antibody (Catalog # IC2274P, filled histogram) or isotype control antibody (Catalog # IC006P, 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.|
The Interleukin 17 (IL-17) family proteins, comprising 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 (1, 2, 6). With the exception of IL-17B, which exists as a non‑covalently linked dimer, all IL-17 family members are disulfide-linked dimers (3). IL-17 family proteins are pro-inflammatory cytokines that induce local cytokine production and are involved in the regulation of immune functions (1, 2, 6). Five receptors (IL-17 RA through RE), which are activated by IL-17 family members, have been identified. Mouse IL-17D is synthesized as a 205 amino acid (aa) precursor that contains a putative 24 aa signal peptide and a 181 aa mature segment. The mature region contains two potential N-linked glycosylation sites and eight cysteines, four of which are involved in the formation of a modified cysteine-knot motif (5). The molecule is reported to exist as a 53 kDa disulfide-linked homodimer (2, 5). Given that its predicted homodimeric molecular weight is 40 kDa, the molecule is presumably glycosylated. In the mature region, mouse IL-17D is 88% aa identical to human IL-17D. There is less than 30% aa identity between mouse IL-17D and other members of the mouse IL-17 family. IL-17D is expressed in skeletal muscle, adipose tissue, fetal liver, and heart, plus resting CD4+ T cells and CD19+ B cells (1). IL-17D is known to induce the production of IL-8, IL-6 and GM-CSF (5).