Slide 8 well chamber slide

All cells were grown on Corning^® Primaria™ 24-well cell culture plates (VWR International, Spånga, Sweden), µ-Slide 8 well chamber slides (Ibidi, Gräfelfing, Germany) or multielectrode array plates (Multichannel Systems, Reutlingen, Germany). Three time points were chosen as illustrative of iPSC, NPC and neuron stages. For infection of iPSCs, the cells were passaged using EDTA (Thermo Fisher Scientific, Waltham, MA, USA) and replated onto Geltrex (Thermo Fisher Scientific, Waltham, MA, USA)-coated plates. For infection of NPCs, cells differentiated for 30 days were passaged using accutase and replated onto laminin-coated plates. For infection of neurons, NPCs were passaged on day 35 of differentiation using accutase and replated onto poly-l-ornithine/laminin-coated plates at a density of 50,000 cells/cm² and differentiated to cortical neurons in NMM for another 55 days.

For immunofluorescence staining of cleaved caspase-3 protein, co-cultures were plated in µ-Slide 8 Well chamber slides (ibidi) on d0. Cleaved caspase-3 staining was performed on d1. For this, samples were fixed for 30-40 min with 4 % paraformaldehyde in PBS at RT, washed with PBS, permeabilized for 10 min with 0.1 % Triton X-100 (Sigma-Aldrich) in PBS at RT and blocked with histobuffer (10 % FCS, 5 % BSA in PBS) for 1 h at RT. Organoids were stained with anti-cleaved Caspase 3 AF648 (BD Biosciences) and anti-EpCAM-AF488 (BioLegend) diluted 1:100 and 1:200, respectively, in histobuffer overnight at 4 °C. The next day, the samples were washed three times with PBS. Nuclei were counterstained for 5 min with Hoechst 33342 (Invitrogen) diluted 1:10000 in PBS. After washing one time with A.d., the slides were mounted with Mowiol 4-88 (Carl Roth). Images were taken with a Leica TCS SP5 II confocal microscope using a 63X objective.

Adherent cells (GOT1, P-STS, QGP-1 and BON-1) were grown on µ-slide 8-well chamber slides (ibidi, Martinsried, Germany) for 3 days and fixed in 4% buffered formaldehyde in PBS. Cells growing in suspensions (KRJ-I, L-STS, and H-STS) were spun onto microscope slides with a Cytospin 2 cytocentrifuge (Shandon, UK) before fixation. The slides were incubated with primary antibodies for 1 h at room temperature in 1% BSA and 0.2% Triton X-100 in PBS and for 1 h with secondary antibodies conjugated to Alexa Fluor 594 (rabbit anti-mouse, cat. no. A11062 or goat anti-rabbit, cat. no. A11037; ThermoFisher) or to FITC (rabbit anti-goat, cat. no. F0250; Dako). All experiments included negative controls wherein the primary antibody was omitted. The fluorescent cells were analysed using a Zeiss LSM 700 confocal microscope and Zen black software was supplied by the manufacturer. Images were captured using a Zeiss LSM 700 inverted microscope with a 63× oil immersion objective.

Synchronized ring-stage parasites were stained with Glucose Uptake Probe-Green (Dojindo, Kumamoto, Japan) and 600 nM MitoTracker Deep Red (ThermoFisher Scientific) for 1 h at 37°C, after which the cells were washed with phenol red-free RPMI containing Washing and Imaging Solution (Dojindo). Cells were dispensed at 0.06% hematocrit to µ-Slide 8 well chamber slides (ibidi, Gräfelfing, Germany) coated with 0.01% poly-L-lysine (Sigma-Aldrich). Prior to imaging, Hoechst 33342 was added to the wells at a final concentration of 2 µM. Cells were imaged within 1 h by Zeiss LSM 780 with the same detector settings, and images were randomly collected by switching every five images between parasites in the presence of TMP or DMSO. The captured images were processed and analyzed using ImageJ. Mean fluorescence intensity (MFI) was measured by the fluorescence intensity attributed to the parasite (red circle as determined by localization with Hoechst/MitoTracker [blue/red] localization) and the erythrocyte (white circle), respectively, as shown in Supplementary Figure 3. Background MFI in the erythrocyte was subsequently subtracted from the MFI in the parasite to provide the change in MFI (ΔMFI).

In all cases, cells were seeded (1.5 × 10⁴ /well) in a sterile µ-Slide 8 well chamber slide (Ibidi) and left to adhere over-night. Imaging was performed on inverted confocal laser-scanning microscope.

ImoDCs and DCP-001, labelled with VPD450 and CFSE, respectively, as described for the uptake assay, were cultured in a 1:1 ratio on a µ-Slide 8 Well chamber slide (ibidi, Gräfelfing, Germany) for 4 h. Cells were then fixed in 4% paraformaldehyde for 10 min at 4 °C. Rhodamin pallodine (1:400, Thermo Fisher Scientific) was added to the cells for further incubation at room temperature in the dark for 2 h. For imaging of the skin crawl-out cells, cells were additionally counterstained with DAPI 1:10,000 (Agilent Technologies, Santa Clara, CA, USA) for 10 min. After washing with PBS once, images were captured using a confocal microscope (Leica SP8 STED, Leica microsystems, Wetzlar, Germany). Fluorescence intensity was measured using ImageJ software version 1.8.0 (NIH, Bethesda, MD, USA).

PC3pip and PC3flu cells were seeded in µ-Slide 8-Well Chamber Slide (ibidi GmbH, Munich, Germany) at 2000 cells/well. When cells grew to 70% confluency, PSMA-1-VcMMAE-Cy5.5 or PSMA-1-McMMAE-Cy5.5 conjugations were added at 50 nM and incubated at 37 °C for 4 h. Cells were washed with PBS and stained with DAPI and LysoOrange (Abcam Inc., Cambridge, UK) for 30 min at 37 °C and 5% CO₂, after which they were washed again with PBS and fresh medium added. Selectivity was determined by including 20-fold excess of the PSMA-1 ligand. The uptake and localization of the drug conjugates were visualized under a confocal microscope (Leica Biosystem, Wetzlar, Germany) at 40×.