Mmo 202nd

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.

A typical experiment setup is shown in Fig. 5. A pressure chamber (specific structure and dimensions are shown in Fig. 6) is connected to a syringe pump (NE-1000, New Era Pump Systems, Inc., 10 ml syringe, B-D Company) that increases or decreases the pressure inside the pressure chamber. A pressure transducer (PX409, Omega Engineering, Inc.) measures the gauge pressure inside the pressure chamber. The pipette pressure probe back-connected with the pressure chamber is installed on a micromanipulator (MMO-202ND, Narishige International USA, Inc.) attached to a microscope (Axiovert 200, Carl Zeiss, Inc.). The pipette probe is pre-loaded with mineral oil. The oil/medium interface in the probe is recorded with a camera (GRAS 20S4M, Point Grey, Inc.) for later image analysis to estimate the curvature of the interface.Figure 5

Experimental setup for pressure measurement. 1. Pressure chamber; 2. Air pump; 3. Pressure transducer; 4. Computer for collecting data; 5. Camera; 6. Object in interest; 7. Pipette pressure probe; 8. Pressure relief valve.

Figure 6

Stucture and dimensions of pressure chamber (units are millimeter, unless indicated). The assemble of bottom plate of chamber is indicated in the cross section A-A of the top part of chamber. The (a) Luer-to-tube connector is locked on the chamber by a (b) specially-designed screw manufactured by a 3D printer.

For basolateral DON exposure, DON (final conc. 1 µM; Sigma–Aldrich Co. LLC, St. Louis, MO, USA) was added to the enteroid culture medium. In contrast, luminal DON exposure was conducted through DON microinjection (28.3 µM in DPBS) into enteroids. Enteroid lumen microinjection and volume measurement of enteroid lumen and microinjection were done as previously described [20 (link)]. The final concentration was equivalent to 1 µM in microinjected enteroids (Table S1). Briefly, the tip of the needle (Femtotips II; Eppendorf AG, Hamburg, Germany) was broken off and its inner diameter was adjusted to 3–4 µm. The 28.3-µM DON solution (final conc. 1 µM) was loaded into the needle using a microloader tip (Eppendorf AG, Hamburg, Germany). Microinjection was performed using a microinjector (FemtoJet^® 4i; Eppendorf AG) and manipulators (MM-92 and MMO-202ND; Narishige Scientific Instrument Lab, Tokyo, Japan) under a microscope (Olympus Co., Tokyo, Japan). Enteroid lumen was stained with 1 µM rhodamine123 (Rh123) for 3 h (Figure S1B,C) to measure its volume. Likewise, DPBS was microinjected into the enteroids to test the effects of microinjection itself (described as “PBS injection” in the figures). For the experiment to test dextran permeability, DPBS was microinjected into the enteroids in the basolateral DON exposure group before exposure in view of the effects of microinjection itself.