Nunc lab tek 8 well chamber slide

Vero cells (80,000 cells per well) were plated in Nunc LabTek 8-well Chamber Slides (Thermo Fisher Scientific) in DMEM supplemented with 9% FBS overnight. Once confluent, 5-fold serial dilutions of each sample were prepared in DMEM supplemented with 1% FBS, and 100 μl of each serial dilution was added to each well in duplicate. After 2 hours, overlay medium (DMEM supplemented with 1% FBS and 1.6% carboxy methylcellulose) was added to the cells, and the cultures were incubated for a further 48 hours at 37°C and 5% CO₂. Cells were washed twice in PBS and fixed for 15 min in 100% methanol. Cells were imaged on an Olympus VS120 Slidescanner microscope (Olympus, Japan) and fluorescent plaques counted using FIJI imaging software [26 (link)].

The experiments were performed as described in Bleicken et al (2013a (link)). Cyt c₄₈₈, APC, and the proteins of interest (20 nM BAX, 100 nM DRP1, 20 nM cBID) were mixed in Nunc™ Lab‐Tek™ 8‐well chamber slides (Thermo Scientific) with GTPase buffer (20 mM of HEPES pH 7.4, 150 mM of KCl, and 1 mM of MgCl₂) and 0.1 mM of GTP. Afterwards, 70 µl of the GUVs suspension was added to get a final volume of 300 µl. Confocal imaging was performed after 60 min incubation at room temperature. Images were processed with a homemade analysis software (Hermann et al, 2014 (link)) detecting the filling and the size of each GUVs. Per sample, well 100–200 GUVs were analyzed. The experiment was performed four times and the results are presented as mean ± SD. Levels of significance were determined by paired two‐tailed Student's t‐test and a confidence level of greater than 95% (P < 0.05) was used to establish statistical significance.

Matrix degradation by primary lung macrophages was measured using the QCM Gelatin Invadopodia Assay (EMD Millipore) according to the manufacturer’s instructions. Lung macrophages were plated at 1.5 × 10⁵ in Nunc Labtek 8-well chamber slides (Thermo Fisher Scientific). Cell induced degradation of fluorescent gelatin was quantified as either the percent of degradation area per cell or the percentage of degradation area per total cell area. Data are displayed as a relative percentage normalised to poly(I:C), with poly(I:C) set to 100%. Analysis was performed according to manufactures instructions using Fiji ImageJ (http://imagej.net/Fiji/Downloads). DAPI and phalloidin signal was thresholded for high intensities, and then analysed as particles to provide a cell (nuclear) count or measurement of cell area. For quantification of the area of degradation Cy3-gelatin signal was thresholded for low intensities, then analysed as particles. Images were obtained using a Leica DM IL LED inverted microscope using a 4×/0.1 Hi-Plan objective and captured using a Leica DFC3000 G camera. Specific band pass filter sets for DAPI, FITC and Texas Red were used to prevent bleed through from one channel to the next.

Purified exosomes were labeled with PKH26 (Sigma-Aldrich), according to the manufacturer’s protocol with minor modifications. Briefly, 1 μL of PKH26 was added to 100 μg of OSCC-derived exosome pellets in a total volume of 400 μL Diluent C and incubated for 5 min at room temperature. The labeling reaction was stopped by adding an equal volume of 1% BSA. Labeled exosomes were ultra-centrifuged at 10,000 × g for 60 min at 4°C. The supernatant was then removed and the pellet was re-suspended in 20 μL PBS. OSCC cells (1 × 10⁴ cells/well) were cultured in Nunc Lab Tek 8-well chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h and pretreated with or without 10 μg/mL heparin for 1 h. Cells were then incubated with 100 μg PKH26-labeled exosomes in the presence or absence of 10 μg/mL heparin for 1, 4, 8, and 16 h at 37°C with 5% CO₂. After incubation, cells were washed twice with PBS and fixed with 200 μL Fixing Solution (Cell Biolabs, San Diego, CA, USA) for 10 min at room temperature. The cells were washed twice with PBS, 200 μL of DAPI solution were added (Cell Biolabs), and the cells were incubated for 15 min at room temperature. Cellular uptake of OSCC-derived exosomes was observed under a confocal laser microscope.

The presence of Wolbachia in cells was also validated through fluorescent in situ hybridization (FISH) at every passage of the virus evolution experiment. Aag-2, Aag-2Tet, and Aag-2wMel cells were seeded onto Nunc Lab-Tek 8-well chamber slides (Thermo Fisher Scientific, Waltham, MA, USA). When the cells were confluent, the cell culture medium was removed and cells were fixed, hybridized with rhodamine-labeled probes specific to Wolbachia 16s rRNA and DAPI, then mounted as per previously published methods (Moreira et al. 2009 (link)). Images were captured using a Leica DM compound fluorescent microscope with a Leica DC300 camera through the appropriate channels, keeping exposure times uniform across samples.

For the confocal microscopy analysis, the biofilm formation was performed on Nunc™ Lab-Tek^® 8-well Chamber Slides (n° 177445; Thermo Scientific, Ottawa, ON, Canada) in Marine Broth medium at 8 and 4°C for 72 h while at −2°C for 96 h. All the microscopic observations and image acquisitions were performed with a confocal laser scanning microscope (CLSM) (LSM700-Zeiss, Germany) equipped with an Ar laser (488 nm), and a He-Ne laser (555 nm). The biofilm observed by CLSM was formed at the bottom of the well.