Gene Therapy for Epilepsies - Gene Delivery and Therapy for Neurological Disorders - page 249

4. While swirling, add equal volume of 2× HeBS buffer (i.e.,
1,000 μl per each 15-cm dish) to the CaCl 2 /DNA mix in the
50-ml conical tube. Leave the mixture for 2 min. A very fine
white precipitate should form. This can be detected visually by
looking at the writing on the tube, which would become a lit-
tle blurry.
5. Take the dishes out from the incubator and quickly but gently
add the transfection mix (2 ml per dish) dropwise in a circular
motion around the dish. Swirl the dish gently to mix and then
return the dish to the incubator.
6. Approximately 12-16 h following transfection, remove the
media and replace with 20 ml of fresh DMEM/10% FBS.
7. 60-72 h after transfection, remove media from cells and
discard.
8. Wash the cells in 20 ml pre-warmed 1× PBS. Carefully add the
1× PBS solution from the side of the dish to avoid directly
flushing against and thereby detach the cells. Swirl the solution
around the dish and then discard the solution.
9. Add 10 ml of pre-warmed 1× PBS to each dish. Detach cells by
using a cell scraper and collect the cell + medium suspension in
50-ml disposable polypropylene conical tubes. If collecting
from multiple dishes, the suspension can be pooled to reduce
the number of conical tubes used, e.g., for five dishes, pool the
suspension and split into two tubes containing ~25 ml each.
10. Pellet cells at 500 × g at 4 °C for 10 min. Discard the
supernatant.
11. Resuspend the cells with lysis buffer (80 mM NaCl, 50 mM
Tris, 1 mM MgCl 2 , pH 8.5). Use 5 ml of lysis buffer per dish,
i.e., use 12.5 ml of lysis buffer for a tube of 25 ml cell
suspension.
12. To lyse the cells, freeze/thaw the lysate 3-4 times on a dry
ice/ethanol bath and a 37 °C water bath. It is important to
minimize disturbing the tubes during the freeze/thaw proce-
dure to help clear the lysate faster.
13. Add benzonase to a final concentration of 50 units/ml. Mix
the tube thoroughly and incubate the lysate at 37 °C for
30 min.
14. Centrifuge the lysate at 4,000 × g for 30 min at 4 °C to remove
the cell debris. Transfer the clarified lysate containing AAV par-
ticles into a new conical tube.
15. Transfer 15 ml of clarified lysate to a 40 ml OptiSeal ultracen-
trifuge tube.
16. Prepare the iodixanol gradient solutions with 10× PBS/10 mM
MgCl 2 /25 mM KCl as detailed in the notes below.