35 mm glass bottomed tissue culture dishes

Cells were seeded in 35-mm glass-bottomed tissue culture dishes (MatTek Corp.) prior to infection with an adenoviral vector expressing either the mitochondrially targeted (mFAP), or ER targeted (ER-FAP) fluorogen activating protein⁴⁴ . At 48 hrs following transfection, cells were pre-stained with 10 μM liperfluo (green) for 30 min before cells were treated with 100 nM RSL3. Before imaging, cells were also loaded with 5 nM malachite green to reveal the mFAP transgene (thereby defining the mitochondria or ER compartments⁴⁵ . All images were acquired using a Nikon Ti inverted microscope (Nikon Inc.) equipped with a 60X 1.49NA oil optic, SpectraX diode based light source (Lumencor), Chroma Technology Inc. filer sets, and an ORCA-Flash4.0 V2 digital cMOS camera (Hamamatsu photonic K.K.). Images were acquired and analyzed using NIS-Elements software (Nikon Inc.)

Cells were seeded in 35 mm glass-bottomed tissue culture dishes (MatTek Corp.) prior to infection with an adenoviral vector expressing mitochondrially targeted fluorogen activating protein (mFAP).^{51 (link)} At 48 h following transfection, cells were pretreated with NAO (25, 50, or 100 nM) and 200 nM tetramethylrhodamine (TMRM) or 200 nM MitoTracker Deep Red (M-22426; Invitrogen) for 30 min. Malachite green (5 nM) was added at 15 min prior to imaging to reveal the mFAP transgene (thereby defining the mitochondria^{52 (link)}). Cells were washed with medium before imaging. All images were acquired using a Nikon Ti inverted microscope (Nikon Inc.) using NIS-Elements Software (Nikon Inc.) and a 60× 1.49NA oil optic and equipped with a SpectraX diode based light source (Lumencor), Chroma Technology Inc. filer sets, and an ORCA-Flash4.0 V2 digital cMOS camera (Hamamatsu).