Journal: Frontiers in Immunology
Article Title: A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It
Figure Lengend Snippet: Staining human neutrophils by anti-IL-17A (AF-317-NA) polyclonal antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) stainings of two FFPE cases of human pustular psoriasis using anti-IL-17A (AF-317-NA) and anti-CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A + cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils, either untreated (top panels) or treated with 5 µM R848 (bottom panels) for 3 h, were stained with anti-IL-17A (AF-317-NA, left panels) and anti-CXCL8 (right panels) Abs. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A) , center/right images in third row in (A) , as well as in (B) , scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF-317-NA immunoblot of lysates from neutrophils either freshly isolated (T 0 , from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17A (rhIL-17A) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.
Article Snippet: Cytokine Production Cytokine concentrations in cell-free supernatants and cell lysates were measured by commercial enzyme-linked immunosorbent (ELISA) kits, specific for: IL-17A (DY317 from R & D systems and 88-7176 from eBioscience), IL-17A/F (88-7117, eBioscience), IL-17B [ABKA2223 from Abnova (Taipei, Taiwan) and ab171344 from Abcam (Cambridge, United Kingdom)], IL-17F (887478, eBioscience), and CXCL8 (Mabtech, Nacka Strand, Sweden).
Techniques: Staining, Immunofluorescence, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Labeling, Marker, Isolation, Incubation, Recombinant, Positive Control