Figure 6
Figure 6. Effect of sialic acid removal on complement activation on endothelial cells incubated in serum. (A) A calcein-release assay was set up to study complement activation, and HUVECs were loaded with calcein and incubated in 15% NHS in the presence of increasing concentrations of the classical complement pathway initiator anti-CD59 antibody. Measurement of supernatant calcein fluorescence (excitation/emission wavelengths of 485/520 nm) showed a dose-dependent response for complement activation. Assessment of an LDH indicator at ex/em values 540/590 nm verified that complement activation did not lead to cell lysis with the anti-CD59 concentrations used. (B) HUVECs were incubated with sialidase or the respective buffer, and removal of sialic acid from cell surfaces was verified by showing decreased binding of MAL II lectin with flow cytometry. Shown is a representative histogram of an assay performed 4 times. (C-E) The role of sialic acid in complement regulation on HUVECs was studied by using the newly developed assay, which detected complement activation as a function of calcein released from the cells. (C) Addition of wt FH19-20 enhanced calcein release, the indicator for complement activation, on untreated but not on sialidase-treated HUVECs. (D) Complement activation on normal HUVECs in the presence of wt and mutant FH19-20 in serum sensitized with anti-CD59 antibody. (E) Complement activation on sialidase-treated HUVECs in the presence of wt and mutant FH19-20 in serum. The assays were performed 5 times. Shown are mean relative fluorescence values (± SD). (F-G) Effect of sialic acid on complement activation on HUVECs was also studied at the level of C3b deposition. (F) Untreated or (G) sialidase-treated HUVECs were incubated in 33% NHS, and FH19-20 was added to interfere with the complement-inhibitory function of FH. Fluorescently labeled C3 was included in the serum to enable later assessment of C3b deposition by flow cytometry. The assays were performed 4 times. Shown are GMFI values (± SD). One-way ANOVA with Tukey’s multiple comparison posttests was performed. **P < .01; ***P < .001.

Effect of sialic acid removal on complement activation on endothelial cells incubated in serum. (A) A calcein-release assay was set up to study complement activation, and HUVECs were loaded with calcein and incubated in 15% NHS in the presence of increasing concentrations of the classical complement pathway initiator anti-CD59 antibody. Measurement of supernatant calcein fluorescence (excitation/emission wavelengths of 485/520 nm) showed a dose-dependent response for complement activation. Assessment of an LDH indicator at ex/em values 540/590 nm verified that complement activation did not lead to cell lysis with the anti-CD59 concentrations used. (B) HUVECs were incubated with sialidase or the respective buffer, and removal of sialic acid from cell surfaces was verified by showing decreased binding of MAL II lectin with flow cytometry. Shown is a representative histogram of an assay performed 4 times. (C-E) The role of sialic acid in complement regulation on HUVECs was studied by using the newly developed assay, which detected complement activation as a function of calcein released from the cells. (C) Addition of wt FH19-20 enhanced calcein release, the indicator for complement activation, on untreated but not on sialidase-treated HUVECs. (D) Complement activation on normal HUVECs in the presence of wt and mutant FH19-20 in serum sensitized with anti-CD59 antibody. (E) Complement activation on sialidase-treated HUVECs in the presence of wt and mutant FH19-20 in serum. The assays were performed 5 times. Shown are mean relative fluorescence values (± SD). (F-G) Effect of sialic acid on complement activation on HUVECs was also studied at the level of C3b deposition. (F) Untreated or (G) sialidase-treated HUVECs were incubated in 33% NHS, and FH19-20 was added to interfere with the complement-inhibitory function of FH. Fluorescently labeled C3 was included in the serum to enable later assessment of C3b deposition by flow cytometry. The assays were performed 4 times. Shown are GMFI values (± SD). One-way ANOVA with Tukey’s multiple comparison posttests was performed. **P < .01; ***P < .001.

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