Alexa fluor 647 conjugated goat anti human kappa f ab 2 antibody

To determine mAb binding capacity to biofilm, wells containing 24 hr biofilm were blocked for 30 min with 4% BSA in PBS. After washing with PBS, wells were incubated with a concentration range of IgG1-mAbs, or Fab fragments when indicated, in PBS-BSA (1%) for 1 hr at 4°C, statically. After washing two times with PBS, samples were further statically incubated for 1 hr at 4°C with APC-conjugated polyclonal goat-anti-human IgG F(ab′)₂ antibody (Jackson ImmunoResearch, 1:500). Fab fragments were detected with Alexa Fluor 647-conjugated goat-anti-human-kappa F(ab′)₂ antibody (Southern Biotech, 1:500). After washing, fluorescence per well was measured using a CLARIOstar plate reader (BMG LABTECH).

Wood46 and LAC ∆spa sbi::Tn biofilm were grown in glass-bottom cellVIEW slides (Greiner Bio-One [543079]) similarly as described above. cellVIEW slides were placed in a humid chamber during incubation to prevent evaporation of growth medium. After 24 hr, wells were gently washed with PBS and fixed for 30 min with cold 1% paraformaldehyde, followed by blocking with 4% BSA in PBS. After washing with PBS, wells were incubated with 66 nM IgG1-mAbs in PBS-BSA (1%) for 1 hr at 4°C, statically. After washing two times with PBS, samples were further statically incubated for 1 hr at 4°C with Alexa Fluor 647-conjugated goat-anti-human-kappa F(ab′)₂ antibody (Southern Biotech, 1:300) and 6 µM Syto9 (Live/Dead BacLight Bacterial Viability Kit; Invitrogen). Z-stacks at three random locations per sample were collected at 0.42 μm intervals using a Leica SP5 confocal microscope with a HCX PL APO CS 63×/1.40–0.60 OIL objective (Leica Microsystems). Syto9 fluorescence was detected by excitation at 488 nm, and emission was collected between 495 nm and 570 nm. Alexa Fluor 647 fluorescence was detected by excitation at 633 nm, and emission was collected between 645 and 720 nm. Image acquisition and processing was performed using Leica LAS AF imaging software (Leica Microsystems).