Transwell Migration Assay

To evaluate the effect of ACh on trophozoite migration, cells were washed and resuspended in plain TYI-S-33 medium without addition of vitamins and serum. 5 × 10⁴ trophozoites were added to the top of a transwell insert containing 8-μm pores (Costar, Cambridge, Massachusetts, USA). Chemoattractant gradients were generated by placing 600 µL of culture SFM containing ACh 1, 0.01, 0.0001, 0.000001 µM, or interferon gamma (IFN-γ), interleukin-8 (IL-8), and 20% ABS in the lower chamber of the migration units. To inhibit chemotaxis, trophozoites were treated with CD. The 24-well plate was kept for 2 h at 37°C and 0.05% CO₂. Cells that migrated to the lower surface of the membrane were fixed with 4% PFA and stained with 1 μg/ml Hoechst 33342 for 20 min. Trophozoite migration was then determined by counting the number of trophozoites that were attached to the bottom of the well with a Carl Zeiss Axiovert 40CFL microscope. Images were processed with the AxioVision 40V 4.6.3.0 software (Göttingen, Germany). Chemotaxis gradient assay was performed to evaluate individual migrating trophozoites, 50 µl of ACh 0.01, 0.0001, or 0.000001 µM were injected into a 0.75% agarose gel, trophozoites were placed around the impregnated ACh agarose gels, and then incubated for 15 min at 37°C. Chemotaxis of trophozoites toward ACh was visualized using phase-contrast video microscopy. Video registers were made with a Carl Zeiss Axiovert 40CFL inverted microscope. Time-lapse videos were generated from 114 spaced frames, with 2 s between each frame acquired for 4-min real-time registers. Each video was processed with AxioVision 40V 4.6.3.0 software. To register random motility of trophozoites in the absence of ACh, SFM was used as a control, additionally, CD was used as a negative control. The data analysis was performed using ImageJ (Wayne Rasband, Nat. Inst. of Health, USA).