Schneider s drosophila medium

Drosophila S2 cells (Drosophila Genome Resource Center; RRID: CVCL_Z232) were maintained at 25 °C in Schneider’s Drosophila Medium (Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 100 unit/ml penicillin and 100 mg/ml streptomycin (Gibco) in 75 cm² cell culture flasks (Corning). For transfections, 30 min prior to transfection, 3 × 10⁶ S2 cells were seeded per well of a 6-well plate (Corning) in 2 ml of serum-containing Schneider’s Drosophila Medium (Gibco). Two hundred to four hundred nanograms of DNA per plasmid were transfected per well using Effectene Transfection Reagent (Qiagen) according to the manufacturer’s protocol. Cells were lysed 48 h after transient transfection. Human embryonic kidney cells (HEK293T) (Crick Cell Services) were cultured in 5% CO₂ atmosphere and 37 °C in DMEM (Dulbecco’s modified Eagle’s medium, SIGMA) supplemented with 10% FBS (fetal bovine serum, Gibco) and penicillin/streptomycin (100 μg/ml, Gibco). Cells were transfected with Lipofectamine ® 2000 (Thermo Fisher) according to the manufacturer instructions. Cells were lysed 48 h after transfection. Cell lines were profiled by STR and tested negative for mycoplasma by the Cell Services Technology Platform at the Francis Crick Institute.

To measure axonal outgrowth during development, flies were reared at 25°C and were dissected in fresh PBS. A minimum of 5 fly brains (10 sLNv projections) per genotype were fixed in 4% formaldehyde and stained with an anti-GFP antibody (Molecular Probes; 1:500), according to standard methods (Ayaz et al., 2008 (link)). For the axonal regrowth analysis after injury, flies were reared at 18°C (to minimize developmental effects), and shifted to 25°C 1 day prior to injury. Whole brain explants on culture plate inserts were prepared as described (Ayaz et al., 2008 (link); Koch and Hassan, 2012 (link)). In brief, culture plate inserts (Milipore) were coated with laminin and polylysine (BD Biosciences). Fly brains were carefully dissected out in a sterile Petri dish containing ice cold Schneider's Drosophila Medium (GIBCO), and transferred to one culture plate insert containing culture medium (10 000 U/ml penicillin, 10 mg/ml streptomycin, 10% Fetal Bovine Serum, and 10 μg/ml insulin in Schneider's Drosophila Medium (GIBCO). sLNv axonal injury was performed using an ultrasonic microchisel controlled by a powered device (Eppendorf) as described (Ayaz et al., 2008 (link); Koch and Hassan, 2012 (link)) and dishes were kept in a humidified incubator at 25°C. Four days later, cultured brains were fixed and immunohistochemical staining was performed as for freshly dissected samples.

Drosophila Schneider 2 (S2) cells were cultured in Schneider’s Drosophila medium (21720024, Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (04-001-1ACS, Bioind, Israel) at 28 °C. S2 cells were split with supplemented medium at a ratio of 1:3 every 3–4 days.
S2 cells were seeded into a six-well plate, and transfection was carried out when the cell growth area reached 70–80% of the well. Lipofectamine 2000 Transfection Reagent (Lipo2000, 11668019, Invitrogen, Waltham, MA, USA), Opti-Minimal Essential Medium (MEM) (31985-062, Gibco, USA), and siRNA were used together to knock down target genes. Transfection was performed according to the following process: Two tubes were prepared to mix reagent, one contained 250 μL Opti-MEM and 15 μL Lipo2000 and the other contained 250 μL Opti-MEM and 15 μL siRNA, and incubated for 5 min after vortexing for 5 s at room temperature, and then mixed with two tubes and incubated for 20 min after vortexing for 5 s at room temperature. The GenePharma Company (Suzhou, China) was responsible for the design and synthesis of the siRNAs. The detailed information of siRNA is listed in Additional file 2: Table S1.