Plate luminometer

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.

Cells were plated in a 96-well clear bottom, black plates at a density of 3,000–5,000 cells/well. To monitor cell growth, aliquots of cell-free conditioned medium (10 μl) were transferred to a white 96-well plate. Gluc activity was determined using FlexStation3 microplate reader (Molecular Device, Sunnyvale, CA, www.moleculardevices.com) under “Flex” mode after adding 100 μl 40 μM of coelenterazine (Nanolight, Pinetop, AZ, www.nanolight.com), which is dissolved in acidified methanol and further diluted in phosphate buffered saline (PBS). To prepare blood samples, 5 μl of blood was withdrawn using a pipette tip from a small incision at the tail tip of conscious mice and immediately mixed with 1 μl of 20 mM EDTA. Gluc activity was then measured using a plate luminometer (Dynex, Richfield, MN, www.dynexproducts.com) after injecting 100 μl of 100 μM coelenterazine and acquiring signal over 10 seconds [28 (link)].

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).