Seakem gtg

A whole lens was held at an angle with the equatorial side down for the live-imaging procedure. An mTmG lens carrying the MLR39-Cre allele was placed in a small hole in the center of an agarose pad that had been prepared by loading 1 ml of 2% agarose (SeaKem GTG, Lonza) in M199 on the surface of 35 mm glass-bottom dish (3970-035, IWAKI). Once the agarose set, 3 ml of M199 with or without 1 ng/ml FGF2 was added to the dish. The lens culture was maintained at 37°C in a 5% CO₂ atmosphere in a stage-top chamber during live-image recording. Y27632 was added to the dish on the microscope stage immediately before recording. Lens fiber cell height was measured by manual tracing using ImageJ/Fiji software.

A mold (Microtissues, Inc., Providence, RI, USA) was used to produce hydrogel plates with 256 wells (16 × 16 wells, 400 μm diameter) as described previously^{23 (link)}. Hot 2.5% agarose solution (SeaKem GTG; Lonza, Basel, Switzerland) in saline was added to the molds and cooled to form a gel. Each agarose hydrogel plate was equilibrated in Dulbecco’s modified Eagle’s medium/F12 (Sigma-Aldrich, St. Louis, MO, USA) overnight before use. A homemade mold (made of polydimethylsiloxane, 1000 wells, 800 μm diameter, 800 μm depth) was also used to prepare agarose microwell plates, and the 1000-well plates were used for long-term culture experiments.

PCR amplifications were performed using Sf9 genomic DNA as a template. DNA was isolated as described above using the Genomic-Tip Kit (QIAGEN) and resuspended in water. PCR amplifications were carried out in a final volume of 50 µl containing 10 µl of 5X Phusion DNA polymerase buffer (HF buffer) (Finnzymes, Finland), 1 µl of 10 mM each dNTP (Biolabs), 0.5 µmoles of each primer (Table S1; Cloning of introns), 1 unit of Phusion DNA Polymerase and 200 ng of Sf9 genomic DNA. After denaturation at 98°C for 2 min, a two-step protocol was used with (i) seven cycles of amplification in the following conditions: denaturation at 98°C for 15 sec, annealing and extension for 3 minutes at 72°C and (ii) thirty cycles of amplification with denaturation at 98°C for 15 sec, annealing and extension for 5 min at 68°C. PCR products were isolated on agarose gels (SeaKem GTG, Lonza) and purified with the QIAquick Gel Extraction Kit (QIAGEN). PCR fragments were then adenylated with Taq Polymerase as described above and cloned in pGEM-T Easy (Promega) for sequencing analysis (Operon Eurofins MWG, Germany).