Low melting agarose

To evaluate the cellular behavior of nasal septum epithelium, we dissected the primary palate from E14.5 and E15.0 K14-GFP embryos for subsequent organ culturing and live imaging (Charoenchaikorn et al., 2009 (link)).
A rolling culture system (Ikemoto Scientific technology, Kanagawa, Japan) was utilized for organ culture, as previously described (Takigawa and Shiota, 2004 (link)). Dissected primary palate specimens were incubated in BGJb medium (Gibco@ Life Technologies) at 37°C in the glass bottle with a rotation speed of 25–30 rpm, in an atmosphere containing 50% O₂, 5% CO₂, and 45% N₂ using a standard incubator for 12 or 24 h. The BGJb medium contains 0.2 mg/L D-Calcium pantothenate and 555.0 mg/L calcium lactate as calcium content.
For live imaging and the quantitative analysis, we removed both palatal shelves (Figure 2B). The nasal septum was cultured in a glass-bottomed dish (Matsunami, Osaka, Japan) with medium containing 0.6% low melting agarose (Wako Osaka, Japan). Explants were cultured and GFP was monitored using an all-in-one fluorescence microscope (BZ-X700, Keyence, Osaka, Japan).

Pregnant mice were euthanized on day 14 of gestation (E14.0) under ketamine (25 mg/kg)/Rompun (8 mg/kg) anesthesia using sterile conditions, and the fetuses were removed from the uterus and placed in BGJb medium (gibco@ Life technologies). Palatal explants were dissected under a dissection microscope as described previously (Charoenchaikorn et al., 2009 (link)). Dissected palatal shelves were cultured in a glass bottom dish (Matsunami, Osaka, Japan) with medium containing 0.6% low melting agarose (Wako Osaka, Japan) (Kim et al., 2015 (link)). The medium was 500 μl of BGJb supplemented with 100 μg/ml penicillin/streptomycin (Invitrogen). Explants were cultured at 37°C with 5% CO₂ using a standard CO₂ incubator for 20 h.