Ritc dextran

The assay was performed as described in el-Fouly et al. [36 (link)] with modification. The confluent cells on 60-mm dishes were washed with PBS containing 1 mM CaCl₂ and immersed in 3 mL of dye cocktail composed of 0.1% Lucifer yellow CH (Sigma-Aldrich) and 0.1% RITC-dextran (Sigma-Aldrich). Several parallel scrape lines were then made with a micropipette tip, and the cells were incubated for 5 min at 37 °C. After rinsed with PBS, the cells dye-coupled with Lucifer yellow were detected under a fluorescence microscope. The cells positive for RITC-dextran were considered to be primarily-scraped but not dye-coupled cells.

To measure fluid-phase endocytosis, 5×10⁵ amoebic transformants were incubated in BI-S-33 medium containing the fluorescent fluid-phase marker RITC dextran (2 mg/ml; Mr = 70,000; Sigma-Aldrich, Japan) at 35 °C for indicated time points. The labelled cells were collected and washed three times with ice-cold PBS. The cell pellets were then suspended in 300 μl of 50 mM Tris-HCl, pH 7.0 containing 1% Triton X-100 and vortexed for 15 s. Fluorescence intensity was measured using fluorometer (F-2500, Hitachi, Japan) at excitation and emission wavelengths of 570 and 610 nm respectively.

hAEpCs were seeded on a chip as described above and cultured on the apical side for 2 days under static and submerged conditions. They were then placed at the air–liquid interface and cultured under static or dynamic conditions for 3 additional days. On day 5, the apical side was washed with PBS with calcium, which was subsequently exchanged with 80 μL of a permeability solution [1 µg mL⁻¹ FITC-sodium (0.4 kDa, Sigma-Aldrich) and 1.5 mg mL⁻¹ RITC-dextran (70 kDa, Sigma-Aldrich) in SAGM™ medium]. The chip was then transferred to the incubator and incubated in static conditions. After 2 h, the apical permeability solution was removed, the apical side was washed once with PBS with calcium, following which the PBS was removed, and the medium in the basal chamber exchanged. The supernatant solution was collected from the outlet well. A 50-µL aliquot of this solution was transferred to a 96-well flat-bottom plate and analyzed with a microplate reader (M1000 Infinite, Tecan) at 460 nm excitation/515 nm emission for FITC-sodium and 553 nm excitation/627 nm for RITC-dextran. The apparent permeability coefficient (Papp) was calculated according to equation (1), dQ/dt being the transport rate and C₀, the initial concentration of the permeability solution tested and A the surface area of the permeability barrier. $$P_{app}=\left(\frac{dQ}{dt}\right)⁎\frac{1}{C_0}⁎\frac{1}{A}$$

The fluid-phase exocytosis assay of E. histolytica trophozoites were performed as described previously [22 (link)]. Briefly, 5X10⁵ amoebic transformants were incubated in BI-S-33 medium containing the fluorescent fluid-phase marker RITC-dextran (2 mg ml^-1; MW = 70 000; Sigma-Aldrich, Japan) at 35°C for 3 h to saturate all endocytic compartments with the marker. The labelled cells were collected, washed and re-suspended in warm marker-free BI-S-33 medium, and incubated at 35°C for 6 h (for gene silenced strain and psAP2 vector control cell line) or 2 h (for overexpressor and GFP vector control cell line). At specific time points (0 h, 3 h and 6 h), cells were collected and washed three times with ice-cold PBS. The cell pellets were then suspended in 300 μl of 50 mM Tris-HCl, pH 7.0 containing 1% Triton-X100 and vortexed for 15 s. Fluorescence intensity was measured using a fluorometer (F-2500, Hitachi, Japan) at excitation and emission wavelengths of 570 and 610 nm respectively.