Bright glo fluc substrate reagent

32 days post-vector injection, mice were sacrificed and organs were harvested for analysis of FLuc levels. Tissues were quickly removed from the animals, and immediately frozen on liquid nitrogen and stored at −80 °C. For FLuc assay, 50 mg of each tissue was placed in 2 ml tubes containing 1.4 mm ceramic beads (Mo Bio Laboratories, Inc., Carlsbad CA) and 500 µL of Mammalian Protein Extraction Reagent (M-PER; Pierce Biotechnology, IL, USA). Tissues were homogenized in a BeadBug microtube homogenizer (Benchmark Scientific, Edison, NJ). Next, tubes were centrifuged for 5 minutes at 300 × g and 20 µL of tissue homogenate was transferred to 96-well white bottom plate and analyzed using 100 µL of Bright-Glo™ FLuc substrate reagent (Promega, WI, USA) and a plate luminometer (Dynex Technologies, VA, USA). A Bradford assay (Bio-Rad, Hercules, CA) was performed to normalize each FLuc value to the total amount of protein in the sample.

SH-SY5Y cells were cultured in EMEM/F12 medium with 10% FBS and 1% penicillin/streptomycin. Cells were seeded at 50,000 cells/well in 96 well plates 24 hours before transduction. 5×10⁷ g.c. of standard ev-AAV9-FLuc or RVG-ev-AAV9-FLuc were preincubated either with heparin (50 µg/mL), or with heparin and RVG (100 µM) for 30 minutes at RT. Cells were transduced for 1 hour at 37°C. FLuc assay was performed 48 hours post-transduction using Bright-Glo™ FLuc substrate reagent (Promega, WI, USA) and a plate luminometer (Dynex Technologies, VA, USA).