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. 2022 Mar 28;204(4):e00035-22. doi: 10.1128/jb.00035-22

FIG 4.

FIG 4

Opa expression state does not affect viability of N. gonorrhoeae in the intracellular or extracellular compartments of neutrophils or the release of neutrophil primary granules. (A and B) N. gonorrhoeae was incubated with adherent, IL-8-treated primary human neutrophils for 1 h. Infected neutrophils were exposed to AF647-coupled soybean lectin to recognize extracellular N. gonorrhoeae and then exposed to BacLight LIVE/DEAD viability dyes in the presence of saponin. The percentage of viable (SYTO9+) N. gonorrhoeae in the intracellular (AF647−) (A) and extracellular (AF647+) (B) compartments was quantified for n ≥ 4 biological replicates. There were no statistical differences among strains in either compartment using one-way ANOVA with Tukey’s multiple-comparison test. (C) Adherent, IL-8-treated neutrophils were exposed to CFSE-labeled N. gonorrhoeae for 60 min. Cells were fixed and stained without permeabilization with rabbit anti-N. gonorrhoeae antibody, followed by AF647-coupled goat anti-rabbit IgG, to label extracellular N. gonorrhoeae. Cells were refixed and exposed to mouse antineutrophil elastase IgG followed by Alexa Fluor 555-coupled goat anti-mouse IgG. The percentage of intracellular (CFSE+ AF647−) N. gonorrhoeae in neutrophil elastase-positive phagosomes was quantified. Results are from n ≥ 3 biological replicates. There were no statistically significant differences by one-way ANOVA with Tukey’s multiple-comparison test. (D) Adherent, IL-8-treated neutrophils were exposed to the indicated strains of N. gonorrhoeae for 60 min. Neutrophils were analyzed for the presence of the primary granule protein CD63 on the cell surface by flow cytometry. Data are presented as the mean fluorescence intensity (MFI) of CD63 and expressed relative to Opaless to account for human subject-intrinsic variability in CD63 expression. Results are from n ≥ 3 biological replicates. Shapes indicate individual matched data points from each experiment. There were no statistically significant differences by one-way ANOVA with Tukey’s multiple-comparison test.