Dmem f12 ham s medium

Transfection was performed using a calcium phosphate co-precipitation method based on previous reports (Dudek et al., 1998 (link); Okada, 2013 ) with slight modifications. In brief, 2 days before transfection, 3 × 10⁶ each of AAV-293 cells was reseeded in four T-150 flasks and cultured to 60–70% confluency in 18 ml of culture medium. One to four hours before transfection, a half volume of the culture medium was replaced with a fresh growth medium. Subsequently, 60 μg each of the plasmid (expression vector, Rep/Cap vector, and pHelper) was mixed well with 6 ml of 0.3 M CaCl₂ solution and then finally combined with an equal amount of 2 × HBS. Three ml each of the final solution was applied to each culture flask. Following a 4-h incubation in a 5% CO₂ incubator, the supernatant was replaced with DMEM/F-12 Ham’s medium (Sigma–Aldrich) containing 2% FBS, 50 mg/ml of streptomycin, and 2mM GlutaMAX I (Thermo Fisher Scientific) and incubated at 37°C in a 10% CO₂ humidified incubator. Cells and culture medium were harvested 2–3 days after transfection, and AAV was purified using the AAV Purification ViraKit (ViraPur LLC) following the manufacturer’s manual. The details of the AAV preparation protocol are available upon request. The virus concentration was quantified using AAVpro^® Titration Kit (Takara Bio) and used at a concentration of ~1.5 × 10¹³ vg/ml.

siRNAs from the first screening, NT siRNA, or anti-Pxn siRNA plus the pVenus-N1 vector were introduced into NBT-L2b cells by the conventional transfection method described above. After 38 h, siRNA-transfected cells (3 × 10⁴ cells) were diluted in 1 ml of DMEM/F12 Ham’s medium (Sigma), supplemented with 10% fetal bovine serum and a mixture of antibiotics, and seeded in wells of a non-treated 24-well microplate (Asahi Glass., Ltd, Tokyo, Japan) that had been coated with a 0.001% solution (w/v) of type I collagen (Research Institute for Functional Peptides, Yamagata, Japan). After a 3-h incubation, the medium was replaced with 1 ml of fresh DMEM/F12 Ham’s medium. The microplate was placed in a Programmable Cellular Image Tracer and incubated for at least 3 h. Fluorescence and phase-contrast snapshot images were captured and phase-contrast time-lapse images were recorded for 6 h at 5-min intervals with the Image Tracer, and analyzed with the ImageJ software. The migration speeds of cells that had been transfected with NT siRNA and the test siRNAs were compared by the Mann-Whitney U-test (statistical significance recognized at P < 0.05, NT versus each siRNA).