Fg 1215

Human tumor cell lines Caco-2 (DSMZ, Braunschweig, Germany) and OE-33, (provided by Dr. Bianca Nitzsche, Charité - Universitätsmedizin Berlin) were grown in tissue-treated petri dishes at 37 °C in a humidified atmosphere containing 5% CO₂. Caco-2 cells were cultured in DMEM Ham’s F12 (FG 4815, Biochrom, Berlin, Germany) medium and OE-33 cell were cultured in RPMI (FG 1215, Biochrom) medium. Bot culture media were supplemented with 10% fetal calf serum (Biochrom), 100 units/mL penicillin (Biochrom), and 100 µg/mL streptomycin (Biochrom).
For spheroid formation, Caco-2 cells and OE-33 cells were grown in growth factor reduced Matrigel (354230, Corning) as previously described [4 (link)]. In brief, 60 µL of Matrigel was added to the growth surface of µ-Plate 96 well (ibidi, Martinsried, Germany) and placed in cell culture incubator for 30 min to allow gel formation. The Matrigel was overlaid with cell culture medium and 5 × 10⁴ cells were added. The cells were cultured for 3–4 days to allow spheroid formation.

In-cell NMR samples were prepared following a recently developed electroporation protocol37 . Briefly, A2780 and RCSN-3 cells were grown at 37 °C, 5% CO₂, in T175 culture flasks in RPMI 1640 (Millipore, FG1215) and DMEM-Ham's F-12 (Biochrom, FG4815) growth media, respectively, supplemented with 10% fetal bovine serum (Biochrom, S0615) until 80% confluence. In all, 50–100 × 10⁶ cells were collected for each in-cell NMR sample. Cells were detached with mild trypsin treatment (Biochrom, L2153) and resuspended in 2 ml of electroporation buffer (100 mM sodium phosphate, 15 mM HEPES, 5 mM KCl, 15 mM MgCl₂, 2 mM ATP and 2 mM reduced glutathione, pH 7.0). Fully oxidized, uniformly ¹⁵N isotope- or ¹⁵N methionine-enriched α-Syn (500 μM) was added to electroporation mixtures and aliquots (100 μl) were used for individual electroporations with an Amaxa nucleofector (Lonza) according to the manufacturer's instructions. Cells were re-seeded in 15-cm culture dishes and allowed to recover for 4 h at 37 °C (CO₂ incubator). Cells were washed 3 × with pre-warmed PBS and collected with mild trypsin treatment, washed 2 × with pre-warmed growth media to remove excess trypsin and resuspended in pH-stable Leibovitz's L-15 medium (Gibco, 31415-029) supplemented with 10% D₂O. Finally, cells were transferred to NMR tubes for in-cell NMR experiments.