Fluoview fv1000 confocal laser scanning microscope

Caco-2 cells were grown at 37 °C with 5% CO₂ on 13 mm poly-L-lysine coated glass cover slips (Thermoline Scientific; Wetherill Park, NSW, Australia) in 24-well plates at a concentration of 5 × 10⁵ cells per well for 48 h. Cells were infected with C. concisus UNSWCD at a MOI of 200 for 6 h. As a positive control for autophagosome formation, Caco-2 cells were treated with 50 μM CQD for 14 h prior to commencing the assay. To investigate autophagy inhibition, 5 mM and 10 mM concentrations of 3-MA were used. Caco-2 cells were fixed in 3.7% formaldehyde and cells were then permeabilized using 0.2% Triton-X 100 in PBS for 15 min at room temperature. Autophagosomes were labelled with anti-LC3B mouse antibody (0.5 μg ml⁻¹) (Sigma Aldrich) and C. concisus UNSWCD was labelled with anti-C. concisus rabbit sera (1:30) and incubated at room temperature for 1 h. The secondary antibodies anti-mouse antibody Alexa Fluor 594 (5 μg ml⁻¹) and anti-rabbit antibody Alexa Fluor 488 (5 μg ml⁻¹) (Life technologies) were added for 1 h at room temperature. Specimens were visualized using an Olympus FluoviewTM FV1000 confocal laser scanning microscope (Olympus; North Ryde, NSW, Australia). Co-localization was measured by calculating Pearson’s correlation coefficients (RTotal and Rcoloc values >0.5 are indicative of co-localization) using the Fiji software (http://fiji.sc/Fiji).

Cells (HeLa, CHO-K1, or pgsA-745) were seeded at a density of 2 × 10⁴ cells/well in 500 μl of complete medium in 4-chambered 35-mm glass bottom Cellview cell culture dishes (Greiner Bio-One, Monroe, NC, USA). After culturing for 24 h, medium was replaced with phenol red– and serum-free medium that contained 25 μM pHK or pHK-PAS, and incubated for 2 h. For some experiments, HeLa cells were preincubated for 1 h at 4°C in serum-free DMEM, pretreated for 1 h at 37°C with 10 mM sodium azide and 6 mM 2-deoxy-d-glucose in serum- and glucose-free DMEM, or pretreated for 30 min at 37°C with 10 µM cytochalasin D in serum-free DMEM. After addition of 25 µM pHK_A488 or pHK-PAS_A488, cells were maintained for 2 h at 4°C or in the presence of inhibitors at 37°C. Thirty minutes before imaging, medium was replaced with fresh medium that contained organelle markers (5 μg/ml Hoechst 33342 and 50 nM MitoTracker Red) or vehicle. Finally, immediately before imaging, medium was once again replaced with fresh medium to remove any extracellular markers. Imaging was performed on an Olympus Fluoview FV-1000 confocal laser scanning microscope using a ×63 Plan-Apo/1.3 NA oil immersion objective with differential interference contrast capability. Image processing was done using Fiji image processing software (Fiji/ImageJ, National Institutes of Health, Bethesda, MD, USA) (24 (link)).

Immunofluorescence staining was performed in cells cultured on glass coverslips as described previously (25 (link),40 (link)). After fixation with 2% paraformaldehyde for 30 min at room temperature, the cells were incubated with anti-ASC (1:100; cat. no. sc-514414; mouse), anti-caspase-1 (1:100; cat. no. sc-1218-R; rabbit), anti-E-cadherin (1:200; cat. no. sc-21791; mouse) or anti-NF-κB (1:200; cat. no. sc-8008; mouse) antibodies (all Santa Cruz Biotechnology, Inc.) at 4°C overnight. After washing, the slides were incubated with Alexa Fluor 488-labeled goat anti-rabbit (1:500; cat. no. ab150077; Abcam) or Alexa Fluor 555-labeled anti-mouse secondary antibody (1:500; cat. no. ab150113; Abcam) for 1 h at room temperature and then subjected to examination using a Fluoview FV1000 confocal laser scanning microscope (Olympus Corporation). Images were analyzed and the co-localization coefficients were calculated using Image-Pro^® Plus (version 7.01; Media Cybernetics, Inc.) (43 (link)). Caspase-1 was detected using Alexa Fluor-488 anti-rabbit IgG secondary antibody and ASC, NF-κB or E-cadherin using Alexa Fluor-555 anti-mouse secondary antibody. For the staining of NF-κB, nuclei were counterstained using Nuclear Green DCS1 (cat. no. ab138905; Abcam).

Cells were grown on coverslips and fixed 4 or 7 d postseeding for 20 min in 3.7% paraformaldehyde. Coverslips and paraffin sections were stained with primary antibodies against acetylated α-tubulin (Sigma-Aldrich), γ-tubulin (Sigma-Aldrich), EEA1 (Sigma-Aldrich), Rab11 (US Biologicals), and Alexa Fluor–conjugated secondary antibodies (Life Technologies). To study transferrin uptake, mIMCD3 cells were washed in serum-free medium and incubated in the same medium with Alexa Fluor 546–conjugated transferrin (Life Technologies) at 37°C, followed by fixation in paraformaldehyde. Images and Z-stack shots were acquired on an Olympus FluoView FV1000 confocal laser scanning microscope using an oil immersion UPLSAPO 60×/1.35 numerical aperture (Olympus, Tokyo, Japan) objective with associated software (FV10-ASW). The Z-step size was 1 μM. ImageJ software (National Institutes of Health, Bethesda, MD) was used to process images and quantify cilia. Cilia frequency was determined by counting cilia per cell. A primary cilium was considered if the long acetylated α-tubulin structure was attached to a γ-tubulin spot (acetylated α-tubulin plus γ-tubulin staining) or a long acetylated α-tubulin structure near a nucleus (acetylated α-tubulin and 4′,6-diamidino-2-phenylindole [DAPI] staining).