In pellet culture, 4 × 105 of SW1353 or fibroblast cells were centrifuged (150 g, 5 min) in a 15-ml polypropylene tube (Becton Dickinson) to form a pellet. The pellets were treated with DMEM (1 g/L glucose) containing ITS supplement, 0.2 mm L -ascorbic acid-2-phosphate, 1 mm sodium pyruvate, 0.35 mm L-proline (all Sigma-Aldrich), and 10 ng/ml TGFβ3 (Biolegend, 585802) or BMP2 (sigma H4791). Medium was changed every two or 3 days (Ullah et al., 2012 (link); Futrega et al., 2021 (link)). Alcian blue staining and extraction of aggregates were performed as described (Wehrle et al., 2019 (link)).
In 384-well ULA plate, 3 × 103 cells were seeded to each well with the medium mentioned above. Lentivirus transduction and PI staining were performed as described (Tambe et al., 2019 (link)). RFP or GFP plasmids were obtained from Addgene (#13726 and #13727). Fluorescence images were acquired using an LSM 710 confocal microscope (Zeiss, Germany).
In 384-well ULA plate, 3 × 103 cells were seeded to each well with the medium mentioned above. Lentivirus transduction and PI staining were performed as described (Tambe et al., 2019 (link)). RFP or GFP plasmids were obtained from Addgene (#13726 and #13727). Fluorescence images were acquired using an LSM 710 confocal microscope (Zeiss, Germany).