Pl apo 63x 1.40 oil cs2 objective

For Confocal imaging, U2OS BAX^−/−BAK^−/− cells seeded on coverslips were transfected with (Halo-BOK, GFP-BOK or GFPBOK∆C) and (GFPSEC61 or mCherry-SEC61) and then treated with 10 µM QVD. 16 h after transfection, the cells were incubated with 150 nM MitoTracker Deep Red (Thermo Fisher) and HaloTag TMR Ligand (Promega) (when transfecting with Halo-BOK) for 20 min at 37 °C. The cells were then washed 3 times with fresh media and were fixed using Paraformaldehyde. Imaging was performed on a TCS SP8 confocal laser scanning microscope (Leica Microsystems) equipped with a PL Apo 63x/1.40 Oil CS2 objective and a tunable white light laser (470–670 nm). The signal was acquired with sensitive HyD detectors (Leica Microsystems).
For STED imaging, U2OS BAX^−/−BAK^−/− cells seeded on coverslips were transfected with Halo-BOK and Smac-GFP and then treated with 10 µM QVD. 16 h after transfection, the cells were incubated with 0.3 μM Janelia Fluor 549 HaloTag Ligand (Promega) and 150 nM MitoTracker Deep Red (Thermo Fisher) for 20 min at 37 °C. The cells were then washed 3 times with fresh media and were fixed using Paraformaldehyde. Images were acquired using TCS SP8 gSTED microscope (Leica Microsystems) equiped with HL PL APO 100x/1.40 Oil STED, a tunable white light laser (470–670 nm) and 750 nm depletion laser. The signal was acquired with sensitive HyD detectors (Leica Microsystems).

For measuring the recruitment of BAX after light-induced (optogenetic) BAK activation, cells were transfected with 50 ng pCRY2-mCherry-BAK, 50 ng pCIBN-mCherry-BAK and 50 ng pEGFP-A206K-BAX-C1 (see key resources table). Cells were kept in dark after transfection. The mitochondria were either visualized using MitoTracker™ Deep Red (Thermo Fisher M22426) staining (100 nM) or using transfection with 100 ng mTagBFP2-Tomm20 (see key resources table). Confocal imaging was performed on a TCS SP8 (Leica Microsystems) inverse confocal laser scanning microscope equipped with a PL Apo 63x/1.40 Oil CS2 objective, a 405 nm laser and a tunable white light laser (470 – 670 nm). Fluorescence emission was detected using HyD SMD detectors. Before imaging, the growth medium was changed to 200 μl of phenol-red free DMEM and cells were maintained in a humidified incubator chamber (Ludin IceCube) with 5% CO2. Individual z-stacks were acquired with 2.5 min interval for approx. 60 min. Maximum intensity z-projection was performed using ImageJ and individual time points before and after BAK foci formation were chosen as representative images.