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. 2023 Nov 22;91(12):e00309-23. doi: 10.1128/iai.00309-23

Fig 4.

Fig 4

Alternative assays for complement-mediated functional antibody activity against N. gonorrhoeae. (A) Complement C3 deposition. N. gonorrhoeae strain FA1090 was incubated with 2C7 or HBSS in the absence or presence of the indicated concentrations of C6-depleted (ΔC6) NHS. Bacteria were fixed, stained with a FITC-labeled anti-human C3 antibody, counterstained with DAPI, and processed for flow cytometry. The fluorescence index was calculated as in Fig. 1. One representative experiment is reported. (B and C) Opsonophagocytic assay with differentiated HL-60 cells. N. gonorrhoeae strain FA1090 was labeled with Tag-IT Violet and then incubated with 2C7 or isotype control, followed by exposure to C6-depleted NHS and differentiated HL-60 cells. After 2 h, cells were fixed, stained with anti-CD11b for differentiation and Zombie Green for viability, and processed for imaging flow cytometry. Gating strategy: (i) singlets with size and aspect ratio indicative of intact cells; (ii) focused singlets; (iii) live cells; (iv) CD11b + cells. In (v), the fluorescence intensity of TIV+ N. gonorrhoeae was measured in the CD11b+ gate. (C) The fold increase in opsonophagocytosis of N. gonorrhoeae by dHL-60 cells elicited by 2C7 compared with the buffer control is reported. Bars represent the mean ± SD of eight independent experiments conducted on different days, with each biological replicate as one data point.