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. 2016 Aug 5;10(8):e0004904. doi: 10.1371/journal.pntd.0004904

Fig 2. Comparison of Sias-siglec mediated binding between a virulent strain (AG83+Sias) and avirulent strain (UR6) with macrophages/monocytes.

Fig 2

Sias-siglec mediated binding of AG83+Sias and UR6 with J774A.1 (A) and THP-1 (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. (C) 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. (D) 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.