μ slide 8 well glass bottom chamber slides

After cell sorting, microglia were cultured as previously described^{5 (link)}. For medium conditioning, proliferation and differentiation medium was collected from OL culture after 24 h or 72 h and spun at 600 × g for 10 min to remove cell debris. 0.8 mL (27%) of conditioned medium (CM) was added to 2.2 mL of microglia medium and 1 mL added to each well for stimulation. For PIC condition, PIC was added at the concentration present in the CM directly to microglia media (13.3 μg/mL corresponding to 27% of 50 μg/mL) and at two different dilutions: 4.4 μg/mL and 1.33 μg/mL. Twenty hour after stimulation RNA was extracted.
To evaluate phagocytosis, microglia were plated in μ-Slide 8-Well Glass Bottom chamber slides (Ibidi, BioValley; 100,000 cells/well). Fluorescent latex beads were activated by incubation with 50% FBS for one hour at 37 degrees with frequent agitation. 100 beads/cells were added to microglia after 21 h of stimulation with PIC or the CM. After 3 h cells were washed twice with PBS and then fixed with 4% PFA.

Biofilms of H. pylori SS1 were prepared as described above using BB2; however, for confocal laser scanning microscopy (CLSM), μ-Slide 8-well glass bottom chamber slides (ibidi, Germany) were used instead of 96-well microtiter plates. Three-day-old biofilms were stained with FilmTracer FM 1–43 (Invitrogen), BOBO-3 (Invitrogen), Filmtracer SYPRO Ruby biofilm matrix stain (Invitrogen), or the FilmTracer LIVE/DEAD biofilm viability kit (Invitrogen) according to the manufacturer’s instructions. Stained biofilms were visualized by CLSM with an LSM 5 Pascal laser scanning microscope (Zeiss) and images were acquired using Imaris software (Bitplane). Biomass analysis of biofilm was carried using FM 1–43-stained z-stack images (0.1-μm thickness) obtained by CLSM from randomly selected areas. The biomass of biofilms was determined using COMSTAT (63 (link)).

Bacterial colonies, grown overnight on blood agar plates, were inoculated into 3 ml of 0.45% saline solution (Air Life, Carefusion, CA, USA) to obtain a turbidity of 0.5 ± 0.1 McFarland (McF) corresponding approximately to 1 × 10⁸ colony-forming units (CFU)/ml. Samples were diluted 1:1000 and resuspended in 1 ml of brain heart infusion broth (BHI) in a μ-Slide, 8 well glass bottom chamber slides (Ibidi, Germany). The bacterial suspension was incubated at 37 °C for 24 h to allow biofilm formation. Afterwards, the medium was removed and biofilms were washed with 0.45% saline solution. The samples were stained using the LIVE/DEAD BacLight kit (Life Technologies, New York, NY, USA), according to supplier specifications. Biofilm samples were analyzed using a Zeiss LSM5 Pascal Laser Scan Microscope (Zeiss, Oberkochen, Germany) as described previously [36 (link)].

Colonies of L. crispatus P17631 and L. paracasei I1688, cultivated overnight on blood agar plates, were used to prepare a bacterial suspension. Specifically, the colonies were suspended in 3 ml of 0.45% saline solution (Air Life, Carefusion, CA, USA) until they reached a turbidity of 2.5 ± 0.3 on the McFarland scale, equating to roughly 1 × 10⁸ CFU/ml. This suspension was then diluted at a ratio of 1:1000 and transferred into 1 ml of BHI within μ-Slide, 8-well glass bottom chamber slides (Ibidi, Germany). The bacterial mixture was incubated at 37 °C over 48 h for biofilm development. After this period, the medium was discarded, and the samples were rinsed with 0.45% saline solution. Biofilm cells were then stained using the LIVE/DEAD BacLight kit (Life Technologies, New York, NY, USA), following the manufacturer's guidelines, and examined with an Apotome system (Zeiss, Oberkochen, Germany) connected to an Axio Observer inverted fluorescence microscope (Zeiss). Data were analyzed with the ZEN 3.2 (blue edition) software (Zeiss).

Regulations for the use of Xenopus laevis, as outlined in the Animals Scientific Procedures Act (ASPA) and implemented by the Home Office in the UK, were followed. Xenopus laevis embryos were obtained by in vitro fertilisation and staged according to Nieuwkoop and Faber (1975). Embryos were injected with capped RNA synthesised using SP6 mMessage mMachine kit (Invitrogen). Animal caps assays were as described 57. Dissociated animal caps were plated onto μ‐Slide 8‐well glass bottom chamber slides (#80827, Ibidi) or 12‐well μChamber slides (#81201) coated with 3 μg/ml recombinant human E‐cadherin protein (#8505‐EC‐050; R&D Systems) in 0.7× Marc's Modified Ringers (MMR; 0.1 M NaCl, 2 mM KCl, 1 mM MgSO₄, 2 mM CaCl₂, 5 mM HEPES pH 7.8, 0.1 mM EDTA).

U20S and HeLa cells transfected with control or COMMD4 siRNA were grown in optical glass bottom 96 well plates (Cellvis) or μ-slide 8 well glass bottom chamber slides (ibidi). Following IR treatment, cells were pre-extracted for 5 min with ice-cold extraction buffer (20 mM HEPES (pH 8), 20 mM NaCl, 5 mM MgCl₂, 1 mM ATP, 0.5% NP40), to remove the soluble proteins and processed as previously described^{22 (link)}. Images were taken using a Delta Vision PDV microscope, ×600/1.42 or ×100/1.42 Oil objective (Applied Precision, Inc) or the OMX Blaze deconvolution SIM super-resolution microscope, ×60/1.42 two Oil objective (Applied Precision, Inc). All immunofluorescence figures were assembled using Adobe Photoshop CS6. High content imaging was performed using the IN Cell Analyzer 2200 Imaging System (GE Healthcare Life Sciences). Images were analysed using the IN Cell Investigator software (GE Healthcare Life Sciences) with a minimum of 50 nuclei quantified per independent experiment.