Nunc lab tek 2 chambered coverglass system

HT22 cells were co-transfected with pCMV G-CEPIA1er and CMV-mito-LAR-GECO1.2 for 48 h prior to seeding into Nunc Lab-Tek II chambered coverglass system (ThermoFisher; 155409) at 1 × 10³ cells/chamber and maintained in Ca²⁺-replete media overnight (Thermofisher; 11995). The following day, the cells were pretreated with 1 μM J147 for 1 h before the medium was removed and the cells were washed 3× with Ca²⁺-free HBSS without phenol red (ThermoFisher; 14175) supplemented with 0.1% FBS (ThermoFisher; 160000) immediately before the addition of thapsigargin (Cayman; 10522). Baseline fluorescence was recorded for 30 s prior to thapsigargin addition. 488 and 568 nm lasers were used to excite pCMV G-CEPIA1er and CMV-mito-LAR-GECO1.2, respectively. Z-stack images were acquired every 30 s and imaged on a Zeiss LSM 880 rear port laser scanning confocal and Airyscan FAST microscope. Zen Black and Imaris imaging analysis software were used to trace and render cells in 3D to quantitate changes in total cell fluorescence over time.

Human embryonic kidney 293T cells were plated in an 8‐well Nunc Lab‐Tek II chambered coverglass system (Thermo Fisher Scientific) at a density of 1 × 10⁴ cells and grown to subconfluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mmol·L⁻¹l‐glutamine, 100 U·mL⁻¹ penicillin, 0.1 mg·mL⁻¹ streptomycin, and 10% FBS.
Primary neural cell cultures from cerebral cortices were prepared from embryonic day 20 (E20) Wistar rats 7. Cerebral cortices were cut into small pieces and incubated at 37 °C for 30 min in papain solution (10 units·mL⁻¹ papain in PBS). The neural cells were passed through a 70‐μm cell strainer (Falcon, Oxnard, CA, USA) to remove debris and then plated in a Nunc Lab‐Tek II chambered coverglass system at a final density of 1 × 10⁵ cells and cultured in DMEM containing 5% FBS. The medium was changed to Neurobasal medium (Sigma‐Aldrich, St. Louis, MO, USA) containing B27 supplement and glutamine (Thermo Fisher Scientific) the next day and supplemented with 10 μm cytosine‐β‐d‐arabinofuranoside (Ara‐C) after 3 days. The culture medium was replaced with new culture medium containing Ara‐C every fourth day. Cells were maintained in an incubator with 5% CO₂ for 10 days.
This study was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Kagawa University, Japan (Approval number 15122).