Opera qehs spinning disk microscope

Generation of the hiPSCs lines, imaging obtainment was performed as previously described^{13 (link)}. The human iPSCs line used in this study is A13777 obtained from Gibco. Briefly, the hiPSCs gene-edited with the Rosella construct into the AAVS1 safe harbour were cultured in Essential-8 media (Thermo Fisher cat no. A1517001) in CellCarrier Ultra plates (Perkin Elmer, 6055300). Confocal images were obtained with an Opera QEHS spinning disk microscope (Perkin Elmer) under a 60x water immersion objective ( $NA·=·1.2$ ). DsRed and pHluorin images were acquired simultaneously using two cameras and binning 2.

Confocal images were acquired on an Opera QEHS spinning disk microscope (Perkin Elmer) using a 60x water immersion objective (NA = 1.2). DsRed and pHluorin images were acquired in parallel using two cameras and binning 2. pHluorin was excited with a 488 nm laser and DsRed with a 561 nm laser. A 568 dichroic mirror was used to deviate the emitted light towards the corresponding cameras. pHluorin was detected on camera 1 behind a 520/35 bandpass filter and DsRed on camera 2 behind a 600/40 bandpass filter. For Rosella-LC3, five planes were set with 400 nm z-steps. For ATP5C1-Rosella, eleven planes were set with 400 nm z-steps. Scale of 1 pixel corresponds to 0.2152 µm in all the cases described.