Viral Manipulation of Neural Stem/Precursor Cells - Viral Vector Approaches in Neurobiology and Brain Diseases - page 264

2.4.2 Surgical
Transplantation of NPCs
into the Spinal Cords

Xylazine-ketamine solution

Ketamine and Xylazin (0.1 mg/g and 0.01 mg/g mouse);
Surgery microscope;
Surgical tools
Iridectomy scissor,
Laminectomy forceps,
Dumont forceps and
Tissue forceps.
5 μl Hamilton syringe

McIlwain tissue chopper;
Millicell culture plate;
Culture medium

2.5 Organotypic
Slice Cultures

50 % OPTI-MEM (Invitrogen),
25 % heat inactivated horse serum (Invitrogen) and
25 % Hank's balanced salt solution (Invitrogen) supplemented
with
d -glucose (5 g/l),
Penicillin (100 U/ml) and
Streptomycin (100 μg/ml).
37 °C, 5 % CO 2 humidified incubator;
Laminar flow cabinet;
Manual micromanipulator;
Vibrationless table.

2.6 In Vivo
Transduction of NPCs

Ketamine and Xylazine;
Stereotactic head frame;
30-gauge Hamilton syringe;
Nanoinjector pump;
Polybrene.

3 Methods

3.1 Preparation
and Culture of NPCs

1. Anesthetize the mice (6-8 weeks old, 18-20 g) by an intra-
peritoneal injection of 4 % chloral hydrate (0.1 ml/kg body
weight) and kill them by decapitation. Cut the parietal bones
in a cranial to caudal orientation using microsurgery scissors;
2. Remove the brains from the skull and place them in a Petri
dish containing sterile PBS. The SVZ region to be dissected is