Nunc lab tek 2 chamber slide system

UBOC1, HK2, and SH-SY5Y cells were plated on two-well chamber slides (Nunc Lab-Tek II Chamber Slide system, 154461, Fisher Scientific, Pittsburgh, PA, USA) and incubated at 37 °C for 24 h. Then the cells were treated with 10, 20, or 5 μM of cisplatin or PBS. Twenty-four hours post treatment, the cells were fixed with pre-cold methanol/acetone (1 : 1 (v/v)) mixture at −20 °C for 15 min. The cells were then permeabilized with 0.2% (v/v) Triton X-100 (Fisher Scientific) in PBS for 30 min, blocked (5% (v/v) goat serum, 2% (w/v) bovine serum albumin in PBS) for 1 h, and incubated with primary antibodies (anti-LMO4, sc-22833; anti-nitrotyrosine, sc-32757; both from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for 1 h at room temperature followed by incubation with secondary antibodies (Alexa Fluor 568 donkey anti-mouse, A10037; Alexa Fluor 647 goat anti-rabbit, A21244; both from Life Technologies) and fluorescein phalloidin (F432, Life Technologies) for 1 h at room temperature. The cells were mounted with ProLong Gold antifade reagent containing DAPI (P36935, Life Technologies), examined using the Carl Zeiss Laser Scanning Systems (Zeiss LSM 780, Jena, Germany), and images were captured and analyzed using ZEN Black imaging software (Ziess). Confocal microscopy was performed at the Microscopy, Imaging and Cytometry Resources Core at Wayne State University, School of Medicine.

Abomasum organoids were cultivated in Matrigel™ for 7 days in an 8-well Nunc Lab-Tek II Chamber Slide System (Fisher Scientific), as described above. IntestiCult Growth Medium was removed and replaced with 250 µl of fresh pre-warmed complete IntestiCult Growth Medium. Fifty microliters of IntestiCult Growth Medium containing ~50 PKH26 labelled O. ostertagi were added to each well of organoids and kept at 37°C, 5% CO₂ for 24-48h. After observing successful invasion into organoids, samples were fixed and prepared for immunofluorescent labelling as described above, with the difference of F-acting labelling by Phalloidin-iFluor 488 (ab176756, Abcam, 1:1000). Images of exL3 worms within and exiting organoids were captured using a Zeiss LSM880 Inverted Confocal Microscope and Zeiss Zen Black operating software. Light microscopy of O. ostertagi within organoids was performed to assess the length of time worms colonised the organoid lumen. After adding exL3 into wells containing organoids, samples were checked hourly to capture the earliest invasion and selected organoids were imaged overnight at a frequency of 10 minutes/image. Time-lapse microscopy was performed in an environmental control chamber at 37°C, 5% CO₂ on a Zeiss Axiovert 200M microscope using Axiovision as operating software.