Syto 61

Biofilm formation examined by CLSM was performed as described previously with minor modifications (Takenaka et al., 2001 (link)). Briefly, overnight culture of PA1, PA1RG, and PA1RG/wzy was diluted 1:100 into fresh LB medium, respectively. Then, 2 ml of aliquot was inoculated into the glass-bottom cell culture dish (15 mm in diameter, Nest, China), and incubated at 37°C for 24 h. The biofilm was washed with phosphate-buffered saline (PBS), and fixed with 2.5% glutaraldehyde for 1.5 h. After washing with PBS, the biofilm was labeled by 5 μM fluorescent nucleic acid stain SYTO 61 (Invitrogen, United States) at room temperature for 30 min, followed by 50 μg/ml FITC-labeled concanavalin A (FITC-ConA, Sigma-Aldrich, United States) at 37°C for 5 min. The biofilm was visualized using the confocal laser scanning microscope LSM800 (Zeiss, Germany) with 561 nm excitation and 640 nm emission wavelengths for SYTO 61, and 488 and 537 nm for FITC-ConA, respectively.

MCF-7 cells were seeded overnight into 4 well chamber slides at a density of 1.5 × 10⁴ cells/chamber followed by a 72-hours treatment with or without either 10 μM 8-Cl-Ado or 50 nM rapamycin. AVO were stained by incubating cells in 50 mM MDC (Sigma-Aldrich) in PBS at 37°C for 10 minutes and then washed three times with PBS. Nuclei were counter stained with 5 μM SYTO 61 (Invitrogen) in Tris-buffered saline (25 mM Tris, 150 mM NaCl, pH 7.5) at 37°C for 10 minutes and then washed twice with PBS and immediately analyzed at UT MD Anderson Cancer Center’s Flow Cytometry and Cellular Imaging Core Facility by fluorescence microscopy using an inverted microscope (Olympus 1X71, Melville, NY) equipped with a filter system (excitation filter: 350/50 nm, emission filter: 528/38 nm). Images were obtained with a Hamamatsu Orca II ER camera (Hamamatsu, Japan) and processed using the program Slidebook (3I, Denver, CO).

Single-species biofilms of E. coli and P. putida that developed after 1 day on all tested surfaces were observed using a 10× dry objective (Leica HC PLAN APO CS) in an inverted microscope Leica DMI6000-CS (Leica Microsystems, Wetzlar, Germany). E. coli cells were pinpointed from the GFP expression, while P. putida biofilms were stained in red with 5 μM SYTO^® 61 (Invitrogen Life Technologies, Alfagene, Portugal), a cell-permeant fluorescent nucleic acid marker. For this reason, E. coli biofilms were observed with a 488 nm argon laser, whereas P. putida biofilm samples were scanned at an excitation wavelength of 633 nm (helium-neon laser) [36 (link)]. A minimum of six stacks of horizontal plane images (512 × 512 pixels, corresponding to 1550 × 1550 µm) with a $z$ -step of 1 μm were acquired for each sample.
Three-dimensional (3D) projections of biofilm structures were reconstructed using the “Easy 3D” tool of IMARIS 9.1 software (Bitplane, Zurich, Switzerland) directly from the CLSM acquisitions. The plug-in COMSTAT2 associated with the ImageJ software was used to determine the biovolume (µm³/µm²) and biofilm thickness (μm) [80 (link)].

Asexual parasite cultures were initiated at 1% parasitemia and 1% hematocrit in triplicate. Parasitemia was measured by flow cytometer (BD FACSCanto) every 48 h by staining with SYTO-61 (Invitrogen). Identical subculturing was performed on wild-type and gene-disrupted parasites to prevent overgrowth. Flow cytometry data were analyzed using FlowJo™ (10.6.0) software. Synchronous gametocytes were produced by the addition of spent media and 62.5 mM N-acetyl-D glucosamine as previously described (60 (link), 61 (link)).

Biofilm membranes were produced according to the method described above. Biofilms were placed in separate wells of a 12-well plate and imaged with a brightfield microscope to quantify initial biofilm areas. Biofilm membranes were then subsequently treated with 10 μL of gentamicin and clindamycin cHG+abx and cHG-only (control group) for 12 hours at culture conditions. MRSA and C. perfringens were treated with a 10X MIC concentration of gentamicin (50 mg/L) and clindamycin (10 mg/L), respectively. At 6 hours and 12 hours of treatment, the biofilm membranes were transferred to individual wells in a 12-well plate for imaging. Bacterial biofilm membranes were stained with SYTO 61 (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions and imaged with a KEYENCE BZ-X700 fluorescence microscope (KEYENCE, Osaka, Japan) to quantify bacterial viability. The bacterial density was calculated at 0, 6, and 12 hours into treatment by measuring the area with viable bacteria divided by total area of the initial biofilm (biofilm at 0 hour).