Fig. 1: P. duboscqi and L. longipalpis salivary glands homogenate contains neutrophil chemoattractants.
From: A sand fly salivary protein acts as a neutrophil chemoattractant
a Flow cytometry analysis of in vivo neutrophils recruitment to C57BL6 mice ears. Mice ears were exposed to a single bite of P. duboscqi and neutrophil (Ly6Ghigh/CD11b+/Ly6Cl°w/−) recruitment was analyzed after different time points. b, d Neutrophils were added to the top chamber of a transwell plate and different amounts of the sand fly salivary gland homogenate were added to the bottom chamber. After 3 h, cells that migrated to the bottom chamber were quantified by DNA content. e Neutrophils were added to the top chamber of a transwell plate and ten pairs of P. duboscqi salivary glands were added to the bottom chamber or to both (top and bottom) chambers and run as in b–d. b, e Murine neutrophils were purified by magnetic negative selection from C57BL/6 bone marrow cells. c Human and d canine neutrophils were purified from peripheral blood of healthy donors by density gradient. f–h Validation of sand fly salivary gland homogenate driven human neutrophil recruitment using EZ-TAXIScan assay. N-Formylmethionyl-leucyl-phenylalanine (fMLP) was used as a positive control. f, g Chemotaxis outcomes were analyzed by Imaris. f Chemotaxis mean speed was computed as the total path length divided by time. g Each track is plotted from the central point and shows XY displacement. h Still images show neutrophils at two different time points (T = 0 m and T = 60 m). Red squares show migrated neutrophils at the bottom of the chamber. Data are presented as means ± SEM. a, e–g Cumulative results of two independent experiments. b–d Cumulative results of 3 independent experiments. a n = 8, 11, 13, 13, 5, 11, 8, 12; *P = 0.0343; ***P = 0.0003. b n = 12, 7, 7, 12, 5, 5, 5; *P = 0.017; ****P < 0.0001. c n = 6; *P = 0.044; **P = 0.0026;***P = 0.0001. d n = 3, 2, 3, 3, 2, 2, 3; *P = 0.0389, *P = 0.0312; **P = 0.0022. e n = 8; ***P = 0.0006. f n = 3, 6, 2, *P = 0.0238; P values calculated by two-tailed Mann–Whitney test (a, e, f) or one-way ANOVA (b–d).
