Cell imaging dish

Enteroids at day 3–4 of culture were transferred onto Cell Imaging Dish (eppendorf) at 100 enteroids/dish as same as the above-mentioned method. For S. Typhimurium ΔphoP injection, the culture medium without antibiotics was used. Microinjection was performed while scanning enteroid at 15 frame/sec by confocal microscopy on Stage Top Incubator. The tip of the needle (Femtotips II, eppendorf) was broke off and its inner diameter adjusted to 3–4 μm was used for microinjection. The needle was inserted into enteroid by using Coarse and fine Three-axis Oil Hydraulic Micromanipulator and One-axis Oil Hydraulic Micromanipulator (MN-4, MMO-202ND, MMO-220A, NARISHIGE), and fluorescein, PBS, 1–10 mg/mL LPS from S.Typhimurium and 1 × 10¹⁰ CFU/mL S. Typhimurium ΔphoP were introduced to enteroid lumen by using Pneumatic Microinjector (IM-11–2, NARISHIGE). Fluorescence intensity of enteroid lumen and medium was measured by NIS-Elements AR. After microinjection, percent area granule secretion was calculated for ten Paneth cells in five enteroids for each sample. The delay time of Paneth cell secretion by microinjection was measured as the elapsed time from the start of introducing reagents into the enteroid lumen until the first granule was secreted.

Cells were seeded on a Cell Imaging Dish (Eppendorf) overnight growth, rinsed with PBS, and then fixed with 4% paraformaldehyde for 30 min. Cells were then rinsed with PBS three times and permeabilized with 0.5% Triton X‐100 for 20 min. Cells or slides with formalin‐fixed GBM tissues were blocked with 5% goat serum for 30 min at 37 °C. Primary antibodies were incubated overnight at 4 °C, secondary antibodies were incubated for 1 h at room temperature, and Hoechst was incubated for 30 min. Images were analyzed on an LSM780 confocal system (Zeiss, Jena, Germany). ZEN lite 2012 was used to analyze the expression of the indicated gene and LC3 dots. The fluorescence intensity of 20 random cells was calculated with a polygon tool and logarithmically transformed. The number of LC3 dots was counted in 20 randomly selected cells.

In order to trace the endocytic route, HeLa cells were incubated with Alexa-Fluor 647-conjugated dextran (Molecular Probes) for 16–18 hr. The cells were washed once with 1X PBS and infected with GFP-expressing Salmonella (at an MOI 50:1) and further incubated in a dextran-free medium for the rest of the experiment. Live-cell imaging was initiated at indicated time-points.
For live-cell imaging experiments, cells were seeded on glass-bottom tissue culture treated cell imaging dish (Eppendorf) and infected with either DsRed- or GFP- expressing Salmonella strains (at an MOI 50:1) as described above. Post-infection, imaging dish was loaded into a sealed live-cell imaging chamber (37°C and 5% CO₂) for imaging in DMEM. Time-lapse confocal images were acquired at specified time-points using an LSM 710 confocal microscope with a LCI Plan Neofluar objective 63×/1.3 multi-immersion correction and equipped with a high-resolution microscopy monochrome cooled camera AxioCamMRm Rev. 3 FireWire (D). Image acquisition and adjustments to brightness and contrast was performed by using ZEN Pro 2011 software.