Opera phenix high content imaging system

We used the ROS-ID® Total ROS/Superoxide detection kit (Enzo life ENZ-51010), which includes two fluorescent dyes—total reactive oxygen species (ROS) detection reagent (Green) and Superoxide Detection Reagent (Orange). Neurons were imaged using Opera Phenix high-content imaging system (Perkin Elmer).

On day 47, HP organoids were dissociated with Accumax (Innovative cell technologies, #AM105) to single cells and stained using a LEGENDScreen Human PE Kit (BioLegend, #700001), which contains PE-conjugated 332 surface marker antibodies and 10 isotype controls. This and the following procedures were conducted in low-cell-adhesion V-bottom 96-well plates (1×10⁵ cells/1 surface marker antibody or isotype control/well). After washing via centrifugation, cells were fixed and permeabilized using an IntraStain kit (Dako, #K2311) and incubated with an FITC-conjugated mouse anti-cytokeratin monoclonal antibody (1:30; clone CK3-6H5; Miltenyi Biotec, #130-080-101, RRID: AB_244191), 5 µg/ml Hoechst 33342, and HCS CellMask Deep Red Stain (Invitrogen, #H32721) for 20 min at room temperature (RT). Finally, cells were washed in Cell Staining Buffer (BioLegend, #420201) and transferred to 96-well imaging plates (CellCarrier-96; PerkinElmer, #6005550). Fluorescence images from each well were acquired and analyzed using the Opera Phenix high-content imaging system (PerkinElmer, RRID: SCR_021100) to determine the percentage of cell populations automatically (1700–11200 Hoechst⁺ nuclei were detected per well). CellMask signals were used for segmentation of cell areas. Cells stained with isotype controls were used as negative controls to determine positive staining for surface markers.

Cells were processed as described before (Pfisterer et al., 2017 (link)) with the following changes: Fixed cell samples were automatically stained with 1 μg/ml LD540 (Princeton BioMolecular Research, (Spandl et al., 2009 (link))) and 5 μg/ml DAPI. 3D stacks of optical slices were acquired automatically either with a Nikon Eclipse Ti-E inverted microscope equipped with a 40 × Planfluor objective with NA 0.75 and 1.5 zoom; duplicate wells, each with six image fields per patient, or with a PerkinElmer Opera Phenix High Content Imaging system with a 63x water immersion objective, NA 1.15; duplicate wells, each with 14, 16 (two wells combined) or 24 (two wells combined) image fields. Image stacks were automatically deconvolved either with Huygens software (Scientific Volume Imaging, b.v.) or a custom-made Python tool based on the open-source tools PSF generator (Kirshner et al., 2013 (link)) and deconvolution lab (Sage et al., 2017 ). Maximum intensity projections were made from the deconvolved image stacks with custom Python tools. Automated quantification of lipid droplets was performed as described previously (Pfisterer et al., 2017 (link); Salo et al., 2019 (link); Vanharanta et al., 2020 (link)).

Cell binding was assessed similarly as described¹² . Briefly, HeLa cells (ATCC, CCL-2), which endogenously express CD98hc, CHO-K1 cells stably expressing cynomolgus CD98hc (ChemPartner, custom order), or CHO parent cells (ChemPartner, item discontinued) were plated in triplicates at 15,000 cells/well in a 96-well Poly-D lysine-coated plate (Fisher Scientific, Perkin Elmer LLC 6055302) and incubated overnight at 37 °C. Cells were treated with ATV^CD98hc:DNP molecules, a positive control anti-CD98hc antibody^{65 (link)}, or anti-DNP negative control antibody for 1 h at 37 °C, fixed with 4% paraformaldehyde, and blocked with 1xPBS containing 5% BSA and 0.3% Triton. Cells were then incubated with anti-human IgG AlexaFluor®488 (1:1000, Jackson ImmunoResearch, 109-545-003), DAPI, and Deep Red Cell Mask (ThermoFisher, C10046). A minimum of 20 field of views were acquired for each replicate at ×40 using the Opera Phenix High Content imaging system (PerkinElmer). Images were analyzed using the Harmony Software (PerkinElmer version 4.9).