Comparison of Sias-siglec mediated binding between a virulent strain (AG83^+Sias) and avirulent strain (UR6) with macrophages/monocytes.

<p>Sias-siglec mediated binding of AG83^+Sias and UR6 with J774A.1 <b>(A)</b> and THP-1 <b>(B)</b> cells were measured by flow cytometry. AG83^+Sias and UR6 were left untreated or treated with sialidase (AG83^-Sias) and labeled with FITC. J774A.1/THP-1 cells were treated with cocktail of anti-siglec antibodies. Cocktail of antibodies were used as inhibitors due to the presence of many siglecs in variable amounts on J774A.1/THP-1 cells. Subsequently, FITC-AG83/UR6 promastigotes were incubated with untreated and treated J774A.1/THP-1 cells at 1:10 ratio and the binding was measured by gating the macrophages in FITC channel. Data were analyzed by CellQuestPro software. Data is represented as mean ± S.D from three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. <b>(C)</b> J774A.1 cells were adhered on the glass coverslip, left untreated or treated with anti-siglec-1,-E,-F antibodies. FITC-AG83^+Sias or sialidase-treated (AG83^-Sias) were incubated with treated and untreated J774A.1 cells at 4°C. Unbound promastigotes were removed; macrophages were stained with phycoerythrin (PE)-anti-CD14 and mounted with mounting media containing DAPI. Images were captured by Andor Spinning Disc Confocal microscope. Green colour represents the FITC-promastigote while red color indicated the surface of macrophages and DAPI stained nucleus were of blue color. <b>(D)</b> J774A.1 cells were adhered on the glass coverslip and treated in the same manner as in Fig. 2B. Cells were infected with FITC-UR6 promastigotes, stained and visualized by confocal microscopy as described in Fig. 2C.