Ethidium homodimer 3

Early passages (between 3 and 6) of hS/PC cells were encapsulated at 3 × 10⁶ cell/mL in HyStem® hydrogel (GS311; BioTime/Ascendance Biotechnology). According to manufacturer's instructions, hydrogels were formed by mixing reconstituted thiol-modified hyaluronic acid (5.9 mM) and polyethylene glycol diacrylate (1.5 mM) at a 4:1 volume ratio, and plated on microscope glass slides fitted with pre-sterilized arrays of 50 μL wells made from laser-cut polydimethylsiloxane (PDMS; Sylgard™ 184; Dow Corning) sheets (Figure S1). Hydrogels were removed from the mold then each transferred into individual wells of a 48-well plate and cultured as described above. A typical hydrogel formed a low-profile cylinder measuring 6 mm in diameter and 2 mm in height. Hydrogels also were formed directly on confocal glass bottom dishes (P50G1.54F; MatTek) for live-cell imaging. 3D cultures were maintained until microstructures reached up to ~50 μm in diameter (between 6 and 16 days), a time when most live-tracking experiments were initiated. Viability assays using Calcein AM (80011-3; Biotium) and Ethidium Homodimer-III (40050; Biotium) in accordance with manufacturer guidelines were performed in cell-laden hydrogel cultures. IMARIS 9.2 software (Bitplane) was used to separate and quantify objects (Movie S1).

HEK293 cells were incubated in 100 µM ManICS1-AM in media containing 5% DMSO for 30 min and then washed in media. Control cells were either untreated (naive), treated with saponin, or treated with DMSO vehicle only. To assess acute toxicity and membrane disruption, a subset of cells was incubated with 4 µM Ethidium Homodimer III (Biotium, Fremont, CA) and assayed for fluorescence at 530 and 620 nm. Higher fluorescence ratio (530/620) indicates increased cell penetrance and intercalation into DNA, indicative of toxicity. To assess long-term viability, an MTT assay (Life Technologies, Carlsbad, CA) was performed. Cells were incubated in the MTT reagent for 4 h at 37 °C to generate formazan crystals, which were then solubilized in sodium dodecylsulfate solution for 2 h at 37 °C and assayed for optical density at 570 nm. Higher absorption indicates higher NAD(P)H-dependent enzymatic activity, indicative of cell health.

Flow cytometry was performed with a LSRII machine (Becton Dickinson) to analyze T-cell developmental and cell death markers from healthy Lck-Dlx5 mice. CD4-APC/Cy7, CD8-PE, CD44-APC/Cy7, CD25-PE, Notch1-Alexa647 and Notch3-APC antibodies were obtained from BioLegend. Annexin V-FITC and ethidium homodimer III were from Biotium. Data were analyzed with FlowJo software.