Glass bottom dish

Hippocampal neuronal culture was performed as previously reported with minor modifications64 . Hippocampi from 18-day-old Sprague Dawley rat embryos were aseptically dissociated and digested with 0.25 w/v% trypsin (Nacalai tesque) for 20 min. After centrifugation, the cells were re-suspended in Neurobasal medium supplemented with 2% NS21 supplement, 0.5 mM glutamine (Invitrogen), penicillin (100 units per ml) and streptomycin (100 μg ml^–1), and were plated on glass coverslip (Matsunami) or glass-bottom dish (Matsunami) coated with poly-D-lysine (Sigma-Aldrich) and laminine (Sigma-Aldrich), and maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The cultured medium was exchanges every 7 days, and the neurons were used at 16–22 DIV. For transfection, after 6–22 DIV, the neurons were transiently transfected with plasmids using Lipofectamine 2,000 according to the manufacturer's instruction. CAM2 reagents tend to be incorporated into dead cells. Thus, we utilized NS21, a serum-free supplement for our neuronal culture.

The membrane-sensitive dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide) was used to detect mitochondrial membrane potential (ΔΨ m) according to the vendor’s instruction with some modifications (10009172; Cayman Chemical Company). Briefly, the JC-1 stock solution was diluted (x40) in the KSOMaa medium (KSOM + JC1). Embryos injected with siRNAs against Gabpb1 or control siRNAs were incubated in KSOM + JC1 for 30 min at 37°C in air containing 5% CO₂. After the incubation, embryos were washed three times in phenol red-free KSOMaa medium containing 0.00025% PVA (KSOM + PVA). Then, embryos were transferred to drops of KSOM + PVA on a glass-bottom dish (Matsunami) and placed in an incubation chamber stage (Tokai Hit) at 37°C under 5% CO₂ in air for live-cell imaging. The fluorescence signals were observed using an LSM800 confocal microscope, equipped with a laser module (405/488/561/640 nm) and GaAsP detector, using the same contrast, brightness, and exposure settings (3 μm interval). For detecting J-aggregates and monomers, the rhodamine and FITC filter settings were used, respectively. Membrane potential is expressed as a ratio of the average rhodamine fluorescence (J-aggregates) relative to the average FITC fluorescence (monomers) in embryo areas.

The contraction of isolated HSCs was measured on a solid gel mixture containing 2.0-μm fluorescent latex beads (Sigma Aldrich, #L4530-1ML). The gel mixture was made with rat tail type I collagen (Millipore, Darmstadt, Germany) and 2.65× Dulbecco’s modified Eagles medium (DMEM, Nissui Pharmaceutical, Tokyo, Japan) and adjusted to pH 7 with 0.5 N NaOH. 150 μL of the gel mixture was plated on a 35 mm glass bottom dish (glass diameter, 14 mm; Matsunami Glass, Osaka, Japan) and flattened. The fluorescent latex beads were diluted 4000-fold with MilliQ water and sonicated for 1 min, and 400 μL of the solution was placed on the gel. The final concentration of collagen in the dish was 1 mg/mL. Collagen was then solidified in a 5% CO₂ incubator at 37°C. Isolated HSCs, which were cultured in DMEM supplemented with 10% fetal bovine serum (Biowest, Cat No: S1820-500, Lot No: S08048S1820), 100 units/mL penicillin, and 100 mg/mL streptomycin and incubated at 37°C in a humidified atmosphere with 5% CO₂ for less than 24 h, were seeded on the solid gel mixture 4 h after the gel preparation. For contraction measurement, the dish solution was replaced by HEPES-buffered solution (125 mM NaCl, 5 mM KCl, 1.3 mM MgSO₄·7H₂O, 1.2 mM CaCl₂·2H₂O, 20 mM HEPES, 5.8 mM D-glucose, pH 7.2–7.4).

2 μl of 50 μg/ml fibronectin was placed on a 35 mm diameter glass bottom dish (Matsunami), or 5 μl was placed on a 96-well plate with clear flat bottom wells (Corning, Corning, NY, USA). One hour later, fibronectin was aspirated, and a 2 μl bead of thawed 4 × 10⁴ iPSC-CM suspension was placed on the 35 mm dish or a 5 μl bead of thawed 5 × 10⁴ iPSC-CM suspension was placed on the 96-well plate. After another one-hour incubation, the appropriate volume of media was added gently. The dish or plate was incubated at 37°C, 5% CO₂. The composition of the media was the same on days 7 to 29. Medium was exchanged every 2 to 3 days.

The suspension of Ald-F(F)F (4.3 mM) and NP-Alexa647 (4.0 µM) with/without Phos-MecycC₅ (2.4 mM) and NBD-cycC₆ (4.0 µM) in 100 mM MES, pH 6.0 was heated by a heating gun until dissolving. After cooling to rt, the resultant mixture (18 µL) was transferred to a glass bottom dish (Matsunami) and incubate at rt for 1 h in the presence of water to avoid dryness. To the resultant solution, a solution of O-benzylhydroxylamine (43.2 mM, 1 µL in 100 mM MES, pH 6.0) or buffer was added. After incubation for 1 h, CLSM imaging was conducted.

The suspension of Ald-F(F)F (17.3 mM) and NP-Alexa647 (4.0 µM) with Phos-MecycC₅ (2.4 mM) and NBD-cycC₆ (4.0 µM) in 100 mM MES, pH 6.0 was heated by a heating gun until dissolving. After cooling to rt, the resultant mixture (10 µL) was transferred to a glass bottom dish (Matsunami) before gelation. After incubation at rt for 1 h in the presence of water to avoid dryness, CLSM imaging was conducted. To the resultant hydrogel, a solution of carboxymethoxylamine (208 mM, 1 µL in 100 mM MES, pH 6.0) or buffer was added. After incubation for 4 h, CLSM imaging was conducted. Subsequently, O-benzylhydroxylamine (300 mM, 2.3 µL in 100 mM MES, pH 6.0) or buffer was added to the resulting solution. After incubation at rt for 48 h, CLSM imaging was conducted.