Fluorescent microscopy was used to measure the interaction of NHBA (100 µg/mL) incubated with tCX cells (cultured on glass coverslips to full confluence) at 37°C for 20 minutes. Cells were washed 3 times to remove unbound proteins and fixed in formaldehyde (2.5% for 15 minutes). NHBA-542 (referred to herein as NHBANg)was detected using anti-NHBA (1:1000) [10 (link)] and Alexa Fluor 488–conjugated anti-mouse immunoglobulin G (1:200; Thermo). Cells were counterstained with Alexa Fluor 568 Phalloidin (Thermo) and DAPI. Glass coverslips were mounted on microscope slides using ProLong Gold Antifade Mountant (Thermo), images were captured on a Nikon A1R confocal microscope, and data were analyzed using NIS-Elements software version 1.2.3 (Nikon).
Gonococcal microcolony formation was investigated using tUEC cells incubated with approximately 1 × 106 CFUs at 37°C for 5 hours. Cell monolayers were washed 3 times (with Hanks’ balanced salt solution) to remove nonadherent bacteria and fixed for 30 minutes, and scanning electron microscopy (JCM-5000 NeoScope; JEOL) was performed as described elsewhere [24 ].
Gonococcal microcolony formation was investigated using tUEC cells incubated with approximately 1 × 106 CFUs at 37°C for 5 hours. Cell monolayers were washed 3 times (with Hanks’ balanced salt solution) to remove nonadherent bacteria and fixed for 30 minutes, and scanning electron microscopy (JCM-5000 NeoScope; JEOL) was performed as described elsewhere [24 ].
