Viral Manipulation of Neural Stem/Precursor Cells - Viral Vector Approaches in Neurobiology and Brain Diseases - page 262

2 Materials

2.1 Preparation
and Culture of NPCs

Mice (7-12 weeks old);
Animal anesthetic (4 % chloral hydrate, 0.1 ml/kg body weight);
Dissecting tools

For brain removal : large scissors, small pointed scissors, and
large forceps;
For SVZ dissection : fine-curved forceps, VANNAS Iridectomy
Scissors Straight, Mouse Brain Slicer Matrix 1.0 mm
Coronal Slice Intervals, razor blades.
Dissecting microscope;

NPC digestion Medium

Eagle's basic salt solution (EBSS), without calcium and mag-
nesium, ice cold;
Papain (100 U/50 ml);
Cysteine (200 mg/l);
Ethylenediaminetetraacetic acid (EDTA; 200 mg/l);

37 °C, 5 % CO 2 humidified incubator;
NPC growth medium

NeuroCult ® Proliferation Kit (mouse; Stem cell technologies)
supplemented with
Mouse recombinant epidermal growth factor (EGF, 20 ng/ml);
Mouse recombinant fibroblast growth factor (FGF)-2, 10 ng/ml;
Heparin at a final concentration of 0.0002 %; w/v; equals
2 μg/ml and
Antibiotics to a final concentration of 100 U/ml Penicillin and
100 μg/ml Streptomycin;

Accumax.

2.2 Lentivirus
Production and
Titration

37 °C, 5 % CO 2 humidified incubator;
Hood (Cat2);
Ultracentrifuge, allowing centrifugation at 100,000 × g , 4 °C;
Tissue culture dishes (150 × 25 mm);
Polyallomer ultracentrifuge tubes;
0.22 μm pore-size cellulose acetate filters;
HEK 293 T cells;
Culture medium for HEK293T cells

Iscove's modified Dulbecco medium (IMDM) supplemented
with 10 % of Fetal Bovine Serum (FBS),