Grace s insect culture medium

Dissected PGs were rinsed with Grace's insect culture medium (Gibco) and pre-cultured individually in 100 µl medium in 96-well plates (BD Falcon) at 25°C for 30 min. To evaluate the effect of extracellular Ca²⁺ on ecdysone biosynthesis, lepidopteran saline [17] (link) and lepidopteran saline using NaCl as a replacement for CaCl₂ were used. For the pharmacological analysis, the PG was pre-treated with the inhibitor for 15 min during the pre-culture period. Following this treatment, the PG was cultured in the desired conditions for the desired period (Ecdysone assay: 3 hours; cAMP assay: 30 min; and Protein phosphorylation assay: 15 min). Actinomycin D, H-89 and cycloheximide (CHX) were purchased from Sigma; Rapamycin was purchased from Santa Cruz Biotechnology; and LY294002 was purchased from Merck. H-89 was dissolved in water. The other inhibitors were dissolved in DMSO. A 1% volume of the appropriate inhibitor was added to the culture medium.

Two ovarian cell lines BmN-SWU1 and BmN-SWU2 were isolated from the ovarian tissue of 3-day-old fourth instar B. mori larvae of the 21-872nlw strain. Briefly, B. mori primary ovarian cell cultures were explanted into a 25 cm² flask at 27°C with 2 ml TC-100 insect medium (USBiological, Swampscott, MA, USA) supplemented with 20% (vol/vol) heat-inactivated fetal bovine serum (FBS, GE Healthcare, Piscataway, NJ, USA). Subcultured cell lines were propagated at 27°C in TC-100 insect medium supplemented with 10% FBS. The BmE-SWU1 cell line was derived from B. mori embryonic tissue and cultured in Grace's insect culture medium (GIBCO, Langley, OK, USA) supplemented with 10% FBS. Fresh ovarian tissues were obtained from 3-day-old fourth instar B. mori larvae of the 21-872nlw strain.