Journal: Immunity

Article Title: Engagement of the type I interferon receptor on dendritic cells inhibits T helper 17 cell development: role of intracellular osteopontin.

doi: 10.1016/j.immuni.2008.05.008

Figure Lengend Snippet: Figure 7. Microglia Require Opn-i Expression to Induce Th17 Cells Opn-deficient microglia were infected with lenti-Opn-i or lenti-GFP and incubated 2D2+CD4 T cells and MOG peptide. (A and B) Reconstitution of Opn-deficient microglia with Opn-i rescues IL-17 production by CD4+ 2D2 T cells. (A) IL-17 concentrations in culture supernatants 24 hr after T cell restimulation on day 4 were determined by ELISA in triplicate wells. (B) Concentrations of IL-27p28 in day 4 cultures before T cell restimulation. The amounts of IL-27p28 were determined by ELISA in triplicate wells; empty and filled bars denote supernatants from Opn-i-transfected and GFP-transfected DC, respectively, and data are representative of two experiments. (C) EAE development is inhibited in irradiated Opn-deficient hosts. Total BM cells (1.7 3 107 cells/mouse) from 2D2+ Tg Opn-deficient mice were i.v. trans- ferred to irradiated (800 rads) hosts. Ten weeks later, EAE was induced. Disease development (mean clinical score) and incidence (%) is shown for each group (n = 5). Error bars in disease score denote mean ± SEM and t test values on the last day of observation are shown. Mice displayed >95% engraftment of donor bone marrow according to immunofluorescence with Ly5.2 Ab (donor BM cells were obtained from B6.Ly5.2+ Opn-deficient mice expressing the 2D2 TCR transgene) 10 weeks after reconstitution and before disease induction, as judged by immunofluorescence of peripheral blood cells.

Article Snippet: Antibodies to IL-17 (TC1118H10) and IFN-g (XMG1.2) for flow cytometry were from BD PharMingen; Foxp3 (FJK-16 s) and IL-10 (JES5-16E3) Abs were from eBioscience; IL-27p28 Ab (BAF1834), recombinant proteins for mouse IL-23, IL-6, Flt-3L, and human TGF-b1 were from R&D systems; and recombinant mouse IFN-a was from HyCult.

Techniques: Expressing, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Transfection, Irradiation

Journal: Journal of neuroinflammation

Article Title: The cytokine IL-27 reduces inflammation and protects photoreceptors in a mouse model of retinal degeneration.

doi: 10.1186/s12974-022-02576-x

Figure Lengend Snippet: Fig. 5 IL-27 localization in the retina. a (Upper panels) Immunodetection of IL-27p28 in rd10 mice at age P25. Colocalization of IL-27p28 signal (red) with GFAP-positive processes (green) indicated by asterisks, consistent with expression in Muller glia. Brightness was enhanced in the GFAP panel in order to visualize the processes in the ONL. (Lower panels) Immunodetection of IL-27p28 in primary Muller glia cultures (red). GFAP (green) detection on an adjacent coverslip indicates the purity of the cultures because every cell was GFAP-positive. The “no Ab” panel contains primary Muller glia with no primary antibody at the equivalent exposure settings as the IL-27 image to demonstrate lack of non-specific immunodetection. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. b DNA gel with PCR products showing IL-27 and IL-27Rα amplification in the BV2 mouse microglia cell line, primary Muller glia (MG) and whole retina from rd10 mice

Article Snippet: The slides were washed in PBS, incubated in blocking buffer (10% goat serum in 0.3% Triton X-100/PBS) for 30 min then incubated overnight at 4 °C in primary antibody solution containing anti-Iba1 antibody (Wako, Richmond, VA, 1:200 dilution), anti-IL-27p28 antibody (Novus, Centennial, CO, 1:100 dilution) or anti-GFAP (Invitrogen, 1:400 dilution), diluted in 2% goat serum in 0.3% Triton X-100/ PBS.

Techniques: Immunodetection, Expressing, Amplification