No 1.5 coverglass

To measure cytosolic ROS in live iPSC-CMs, cells were loaded with 5 µM CellROX™ Deep Red Reagent (Molecular Probes, OR, USA) and 2 µM Hoechst 33,342 (Invitrogen) at 37 °C for 30 min, and then washed 3 times with PBS. For mitochondrial morphology and ROS detection, iPSC-CMs were loaded with MitoTracker^® Green FM (Molecular Probes) at 200 nM and MitoSOX™ Red (Molecular Probes) at 5 µM for 15 min at 37 °C, then washed gently 3 times with warm buffer before imaging. For the treatment of maturation medium (MM), iPSC-CMs were switched to MM for 48 h before functional analysis. For the treatment of compounds, N-Acetylcysteine amide (NACA) was used at 5 mM and rapamycin used at 0.5 µM final concentration, respectively. For live cell imaging analysis, all the cells were seeded on Lab-Tek^® II chamber glass slides (Nunc) or glass-bottom 35 mm dishes with 14 mm microwell and No. 1.5 cover glass (MatTek, Ashland, MA, USA).

Cultured primary DRG neurons from thoracic and lumbar (T12–T13, L1–L6) were used for calcium imaging experiments. DRGs were dissected and placed in chilled HBSS solution. The neurons were then digested in collagenase A (1:1; A (Sigma-Aldrich, Cat#10103586001):HBSS (Gibco, Cat#14-170-112)) for 20 min at 37 °C, collagenase D (1:1:10%; D (Sigma-Aldrich, Cat#1188866001):HBSS:papain (Sigma-Aldrich, Cat#10108014001) for 20 min at 37 °C, and then placed in a Trypsin Inhibitor solution (1:1:1; Trypsin (Sigma-Aldrich, Cat#10109886001):BSA: Media) for trituration. Media is DME/F-12 1:1 (1) with 2.50 mM l-Glutamine and 15 mM HEPES buffer (HyClone, Cat#SH30023) supplemented with 10% Fetal Bovine Serum (HyClone, Cat#SH30088.03) and 1% Penicillin Streptomycin (Fisher Scientific, Cat#15070063). After trituration, cells were filtered through a 70 μm cell strainer (Corning, Cat#CLS431751), pelleted, buffer removed, and then resuspended in 200 μL of Media. Plates used were 35 mm Petri dish, 10 mm Microwell, and No. 1.5 cover glass (MatTek Corporation, Cat#P35GC-1.5-C) that were coated in facility with a 2 µg/mL poly-D lysine solution (Sigma-Aldrich, Cat#P0899). Cells were plated in a 200 μL bubble on the center of the plate to rest in an incubator set at 37 °C with 5% CO2 for 2 h before filling the rest of the well with Media. Cells were used the next day for calcium imaging.

Cells were plated on collagen (50 μg/ml) coated 35 mm dishes glass bottom dishes with no. 1.5 coverglass (MatTek corporation), Lipofectamine 3000 was used to transfect cells as per the protocol described by the manufacturer. Briefly, the DNA and lipid reagent were separately diluted in Opti-MEM. Prior to transfection they were combined and added to dishes. Cells were incubated overnight with the DNA-lipid complexes and imaged the next day. Prior to imaging, the media was replaced with warm FluoroBrite DMEM Media (Life Technologies) supplemented with 10% FBS. Cells were imaged on an LSM880 Zeiss inverted microscope outfitted with confocal optics with the 63× oil 1.4 numerical aperture (NA) objective and Airyscan. The environment throughout imaging was controlled at 37°C, 5% CO₂, and 90% humidity.

Cells were plated into 35-mm glass bottom dishes (14 mm, No. 1.5 coverglass; MatTek Corporation) and transfected with siRNAs. The medium was replaced at least 6 h before filming with L-15/10% FBS. Four-dimensional data sets were acquired with a spinning disc confocal system (Yokogawa) equipped with an electron multiplying charge-coupled device camera (iXonEM+; Andor) and a CSU-22 unit (Yokogawa) based on an inverted microscope (TE2000-U; Nikon). Two laser lines (488 and 561 nm) were used for near-simultaneous excitation of GFP and mRFP, and the system was driven by NIS Elements 3.0 software (Nikon). Time-lapse imaging of z stacks with 0.7-μm steps covering the entire volume of the mitotic apparatus were collected every 1–2 min (Supplementary Movies 1–6) or every 2 s (Supplementary Movies 7–9) with a plan-apochromat 1.40 NA × 60 immersion oil objective3 (link). Brightfield microscopy was performed on a Nikon Eclipse TE2000-U microscope driven by NIS Elements 3.0 software with × 63 or × 100 objectives. Time lapse was performed every 30 s, 1 or 2 min, depending on the type of experiment.