Modification of side chains of terminal sialic acids on erythrocyte surface by mild periodate and MTSC. (A) Mild oxidation using sodium periodate (NaIO4) generates aldehydes on sialic acid–containing glycoproteins, followed by direct labeling of aldehydes with a fluorescent tag, FTSC (top, green). A smaller compound, MTSC, replaced FTSC, which would generate the same sialic acid modification without the fluorescein molecule (bottom, blue). (B) By flow cytometry, modification of sialic acid by both FTSC and MTSC was tested for reactivity and competition on the erythrocytes surface. Erythrocytes were treated with sodium periodate (NaIO4) for 20 minutes on ice, followed by the addition of FTSC or MTSC, where noted, for 1 hour at 37°C; n = 2. (C) Sialic acid modification of treated erythrocytes (NaIO4 + MTSC, MTSC only) and untreated erythrocytes (DPBS) were stained with biotinylated SNA lectin, which preferentially binds to sialic acids attached to terminal galactose in α2-6-linkage and was measured by flow cytometry. Isotype control (streptavidin-PE); n = 4. (D) Purified neutrophils were incubated with erythrocytes (DPBS, MTSC only, and NaIO4 + MTSC). Erythrocyte concentration was at 1:50 neutrophil:erythrocyte ratio. MFI of phagosomal ROS production was analyzed using Fc-OxyBURST Green assay reagent at 15 minutes. Statistics were analyzed by ordinary 1-way ANOVA; n = 2. **P < .0072 versus control values considered statistically significant; ***P < .0004.
