Zen 2011 program

One hundred and fifty μL of 4’,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA, USA) solution (25 μg/ml) was applied on the immobilized E. coli cells for 15 min. After washing with PBST three times, the E. coli cells were stained with 150 μL of FITC-PMB solution (1 μg/ml) for 30 min. After washing with PBST three times, the E. coli cells on a glass slide were dried in a desiccator at room temperature for 1 hr. LSM700 confocal laser scanning microscope (CLSM) (Carl Zeiss, Jena, Germany) was used to visualize E. coli cells stained with DAPI and FITC-PMB, respectively. Fluorescence microscopic images were taken with a 40 X objective lens (C-Apochromat 40×/1.20 W Korr M27, Carl Zeiss) and with the filter sets for DAPI (excitation 350 nm, emission 470 nm) and FITC (excitation 490 nm, emission 525 nm), respectively, and processed using the Zen 2011 program (Carl Zeiss).

After the flow cytometric or fluorometric readings were gathered, representative samples of cells were fixed in 3.5% paraformaldehyde solution for 20 min on ice, washed, and stained with the Hoechst 33342 dye (1 μg/ml), for nuclear staining. The slides were mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA) and examined using confocal microscopy.
To confirm localization of bacteria within LAMP1-expressing phagolysosomes, representative fixed cells were permeabilized with 0.05% saponin solution and stained with Alexa Fluor 488-conjugated anti-mouse LAMP1 (expressed on lysosomal structures in cells) antibody (25 μg/ml; Biolegend, San Diego, CA).[36 (link)] Hoechst 33342 dye (1 mg/ml) was then added for nuclear staining. All images were acquired using a 63X oil immersion objective in a Zeiss confocal microscope, and were processed using the Zeiss ZEN 2011 program.

P. aeruginosa biofilms were formed in a drip-flow biofilm reactor (DFR-110, BioSurface, MT, USA). Glass slides were dipped into a petri dish containing 2 ml of P. aeruginosa culture (OD at 595 nm = 1.5) and 18 ml of fresh AB medium to attach cells onto the slides and were incubated at 37°C for 24 h. The slides were then inserted into the drip-flow reactor system. Fresh AB medium with either 0 or 10 μM 6-gingerol was continuously fed into the reactor using a peristaltic pump (Masterflex C/L tubing pumps, Cole-Parmer, IL, USA) at 50 ml·h⁻¹. The reactor operated at 37°C for 24 h. After stopping the feed, the suspended cells on the slide were carefully removed with phosphate-buffered saline (pH 7.2). The biofilm cells were stained with DAPI solution (Carl Roth) for 20 min. The biofilm cells were then observed using CLSM (Carl Zeiss LSM700, Jena, Germany) based on a previous study23 (link). Confocal images of DAPI-stained biofilm cells were observed under blue fluorescence light (excitation wavelength: 350 nm, emission wavelength: 470 nm) using a 40× objective lens to evaluate the height and density of the biofilms (C-Apochromat 40×/1.20 W Korr M27, Carl Zeiss). The observed CLSM images were analyzed by the Zen 2011 program (Carl Zeiss).