Grace s insect cell culture medium

Sf9 cells were cultured in 35 mm² flasks (Nest, Wuxi, China) with Grace’s insect cell culture medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco) at 27 °C. The medium was changed every two days, and the subculture was operated after the growth of the cells to 70–80% of the flask.
A colony of S. frugiperda was derived from a cornfield collection in Huizhou city, Guangdong province, China. Larvae were fed with an artificial diet, while 10% honey water was used as the food for adults. The population was maintained in an incubator with the following parameters: 25 ± 1 °C, 70% relative humidity, and a photoperiod of 12:12 (L:D) h.

High 5 Cell lines (Hi-5) derived from embryos of T. ni, and SL cell lines derived from the ovary of S. litura, were obtained from the Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, Central China Normal University, China. Two cell lines were cultured in Grace’s insect cell culture medium (Gibco, America) and supplemented with 10% fetal calf serum at 27.5 °C. Cells in the logarithmic phase of growth were used in the experiments. All the tested compounds were dissolved in dimethyl sulfoxide (DMSO) and then diluted to the desired concentrations with cell culture medium before use. The concentration of DMSO was kept 0.1% in treated groups. Control cultures were performed in the presence of 0.1% DMSO under the same culture conditions [34 (link)].

Spodoptera frugiperda Sf9 cells were cultivated at 27°C and 5% CO₂ in 3 mL Grace’s insect cell culture medium (Gibco, United States) containing 10% fetal bovine serum (FBS), 0.3% yeast extract, 0.3% lactalbumin hydrolysate, and 0.3% peptone. Chinese hamster ovary (CHO-WTA11) cells were cultured at 37°C and 5% CO₂ in the DMEM/F-12 medium (Invitrogen Life Technologies, Carlsbad, CA, United States) supplemented with 10% FBS, 250 ng/ml fungizone, 100 U/ml of penicillin, and 100 mg/ml of streptomycin.
In order to detect the effect of scopoletin (purity, 95%; Southwest University, Beibei, Chongqing, China) on intracellular free calcium [Ca²⁺]i levels in insect cells, the Sf9 cells were treated with diluted scopoletin at concentrations of 0, 10, 50, and 90 μM for 24 h. The [Ca²⁺]i level in the Sf9 cells was determined by Fura-2/AM fluorescence staining (Cao et al., 2016 (link)). Briefly, the harvested cells were incubated with Fura-2/AM (Beyotime, China) at final concentration of 5 μM at 30°C for 30 min. A fluorescence microscope (Carl Zeiss) was then used to observe the cells. The [Ca²⁺]i level was represented by the mean fluorescence intensity (MFI) after the captured images were analyzed using Image-Pro Plus software (Media Cybernetics).