Ipl 41 insect medium

GFP-expressing bacteria were used to inoculate fifth instar day 2 larvae, which were then incubated at room temperature with food. At 1, 24, 72, and 120 h post-inoculation, hemolymph was collected from the caudal horn and added to a 24-well tissue culture plate, before diluting up to 500 μL with IPL-41 Insect Medium (Sigma-Aldrich, St Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum. The plates were then centrifuged for 5 min at 900 × g and incubated for 15 min at room temperature. After washing twice with PBS, the samples were fixed with 4% paraformaldehyde (Wako, Osaka, Japan) in PBS for 15 min at room temperature. Subsequently, the samples were washed twice with PBS. Fluorescent images were obtained using a FluoView FV100 confocal laser scanning microscope (Olympus).

The selected cell lines were available to the labs participating in this study.
High Five cells (Trichoplusia ni) were maintained at 27.5 °C in IPL-41 Insect Medium (Sigma-Aldrich, Bornem, Belgium), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich), 0.25 µg/mL amphotericin B (Sigma-Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Life Technologies, Merelbeke, Belgium).
TcA cells (Tribolium castaneum) were maintained at 27.5 °C in EX-CELL^® 420 medium (Sigma-Aldrich), supplemented with 10% heat-inactivated FBS (Sigma-Aldrich), 5 µg/mL human insulin (Sigma-Aldrich), 0.25 µg/mL amphotericin B (Sigma-Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Life Technologies, Merelbeke, Belgium).
S2 cells (D. melanogaster) were maintained at 25 °C in Shields and Sang M3 Insect Medium (Sigma-Aldrich), supplemented with 1 g/L yeast extract (Sigma-Aldrich), 2.5 g/L Bacto™ Peptone (BD Biosciences, San Jose, CA, USA), 10% heat-inactivated FBS (Sigma-Aldrich), 0.25 µg/mL amphotericin B (Sigma-Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Life Technologies).

The Bombyx mori ovary-derived cultured cell line BmN4 was a gift from Dr. Kusakabe [16 ]. BmN4 cells were cultured in IPL-41 insect medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) at 25°C in the atmosphere. BmN4 cells were seeded (1 × 10⁵ per well) in 48-well tissue culture plates coated with concanavalin A at 48 h prior to use. Plate coating was performed according to the following steps: the bottoms of the wells were covered with 100 mM concanavalin A and incubated for 2 h at room temperature, before removing the concanavalin A solution, air drying completely, and washing once with PBS.

The BmN cell (Funakoshi Co., Ltd., Japan) was maintained in IPL41 insect medium (Sigma, St. Louis, MO) with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY) at 27°C. The silkworm strains used in this study were provided by the Institute of Genetic Resources at Kyushu University, and the silkworm larvae were reared on mulberry leaves at 24–29°C.

The Bm5 and High Five cell lines were maintained in a complete medium consisting of IPL-41 Insect Medium (Sigma-Aldrich), supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 0.25 μg/ml of amphotericin B (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Life Technologies). The cells were subcultured weekly and maintained at 27.5 °C.
Bm5 and High Five cells were transfected using Escort IV (Sigma-Aldrich), according to manufacturers’ instructions. An optimized volume of the transfection reagent was used, namely 3.7 μl and 15 μl per well of a 24- and 6-well plate, respectively.
In the overexpression experiments, the expression constructs were transfected overnight, at a concentration of 1 μg/ml. In addition, the pBmIE1 helper plasmid encoding the ie-1 gene for B. mori nuclear polyhedrosis virus^{48 (link)} was used, at a concentration of 0.3 μg/ml. In the knockdown experiments, the dsRNA was transfected at a concentration of 2.5 μg/ml. After the transfections, the medium was replaced for the complete medium above mentioned.