Ibitreat chamber slides

Overnight cultures of C. albicans and mCherry-labelled E. coli, GFP-labelled S. aureus, or GFP-labelled MRSA were prepared as outlined above and biofilms were formed in μ-Slide 8 well ibiTreat chamber slides (ibidi, Germany) by inoculating wells with 100 μl C. albicans laboratory strain or C. albicans gcn5Δ/Δ hyphal-deficient mutant (1.0 x 10⁶ cells/ml in RPMI). Wells were incubated for 4 hours to allow initial C. albicans biofilm formation. The biofilm was washed with PBS to remove planktonic cells and RPMI, prior to inoculation with 100 μl mCherry- or GFP-labelled bacteria (5.0 x 10⁶ cells/ml in BHI). Bacteria were allowed to adhere to the C. albicans biofilms for 4 hours to facilitate initial dual-species biofilm formation. Images were acquired by TIRF microscopy (Leica UK) in the epifluorescence mode using a Leica EL6000 external light source for fluorescent images and LED lamp for bright field images. Fluorescent and bright field images were overlaid using LAS-X software (Leica Application suite).

To analyze the subcellular localization of actin, different human macrophage subtypes were cultured in the presence of storage buffer (vehicle control) or 10 μg/ml recombinant human galectin-2 at day 7 on ibitreat chamber slides (Ibidi, Planegg/Martinsried, Germany) at a density of 1 x 10⁶ cells/ml in DMEM complete medium. Macrophages were fixed in 4% PFA in HBSS (Invitrogen) at RT for 10 minutes, and permeabilized with 0.1% Triton X-100 (Merck) in PBS at RT for 5 minutes. Finally, the cells were stained with 0.4 μg/ml phalloidin-tetramethyl rhodamine iso-thiocyanate (TRITC; Sigma-Aldrich) at RT for one hour. Imaging was performed by a confocal laser scanning microscope (Leica TCS SP2 AOBS, Leica Microsystems B.V., Rijswijk, The Netherlands). A total of five randomly selected fields at 63 times original magnification were acquired with Leica confocal software version 2.61 (Leica Microsystems, Wetzlar, Germany).

Cells were seeded 24 h prior to the experiment in μ-slide 8-well ibitreat chamber slides (Cat. 80,826, Ibidi, Germany). Cells were fixed, permeabilized and incubated with the diluted antibodies against ER, PR and HER2 overnight at 4 °C. All antibodies were purchased from Cell Signaling. Secondary FITC antibody (Life technologies) was used to detect antibody fluorescence and counter staining was performed with HOECHST (Life Technologies) to detect nucleus. Slides were imaged using LSM 780 (Zeiss, Germany).

Macrophages and tumor cells were labeled with the fluorescent cell labeling solutions Vybrant DiI and CellTracker Violet (Life Technologies), respectively, by 30 min incubation at 37°C according to the supplied instructions. Macrophages were plated on IbiTreat chamber slides (Ibidi, Martinsried, Germany) with culture inserts. Inserts were then removed, and the slides overlaid with tumor cells in 50 µL Matrigel. The position of individual tumor cells relative to the macrophage border was determined using ImageJ v.1.47 (NIH), and frequency distribution plotted to reflect the spatial distribution of tumor cells relative to the macrophage monolayer.
Live imaging experiments were carried out on an Olympus FV 1000 confocal microscope equipped with a 20× Olympus Plan Achromat Objective, 0.4 NA, 1.2 mm WD objective. The cells were then imaged for 24 h at 37°C with 5% CO₂, with image acquisition every 10 min. In other experiments, an IncuCyte wound maker (Essen BioScience, Ann Arbor, MI, USA) was utilized to remove a defined portion of macrophage monolayers in 96-well plates, and growth of tumor cells in the scratch area of individual wells was imaged with 20 min intervals for 48 h using IncuCyte Zoom live-cell imaging system (Essen BioScience).

2.5–5 × 10⁴ iBMDMs were seeded in 8‐well μ‐slide ibiTreat chamber slides (iBidi). The following day cells were imaged live using the 3i marianas spinning disk confocal microscope with CSU‐W1T2 Yokogawa spinning disk confocal (50 μm disk) coupled to inverted Zeiss Axio observer 7 stand. iBMDMs were imaged before or after treatment with 50 μg/ml DMXAA. Z stack images were acquired with a 0.5 μm z‐step over time. Details for image acquisition can be found in figure legends. Imaris software was used to export 3D movies for visualisation as mp4 files. Single images or image sequences were exported from ImageJ either as maximum intensity projections (MIPs) or a single z‐slice.

Cells were plated in 60 μL of Matrigel in μ-slide eight-well ibiTreat chamber slides (Ibidi, Martinsried, Germany) and overlaid with advanced DMEM/F12 supplemented with Zell Shield antibiotics, 1× B27 and 1× N2 supplement. Cell-derived organoids were observed for 16 days and then they were fixed in situ for 5 min each with −20 °C 100% methanol and 2% formaldehyde in PBS. The chambers were washed twice for 5 min with PBS and once for 5 min with PBS-Tween. The slides were blocked with 3% bovine serum albumin for 30 min and incubated with β-catenin antibody (BD, 1:50) at 4 °C overnight and washed three times for 5 min with PBS-Tween. Organoids were then incubated with Alexa Fluor 488 secondary antibody (Thermo Fisher, 1:200) at 4 °C overnight and were washed three times for 5 min with PBS-Tween. Nuclei were stained with DAPI and serial images were taken using a Zeiss LSM 710 confocal microscope.