Primary Culture of Adult Neural Progenitors - Drugs of Abuse: Neurological Reviews and Protocols - page 389

3. Methods
Methodology is described for tissue preparation ( Subheading 3.1. ), single
cell dissociation/neurosphere culture ( Subheading 3.2. ), and assessment of
progenitor differentiation potential ( Subheading 3.3. ).

3.1. Dissection and Tissue Preparation
This protocol describes the euthanasia procedure ( Subheading 3.1.1. ) and
dissection parameters ( Subheading 3.1.2. ) underlying neurosphere preparation.

3.1.1. Animal Euthanasia
Mice were deeply anesthetized with sodium pentobarbital and decapitated.
Brains were rapidly removed and blocked at approx bregma 1.4 and 0 mm
under a dissecting microscope ( see Note 1 ). Tissue blocks were placed in 10 mL
ice-cold aCSF in 10-cm tissue culture dishes with gentle shaking on an orbital
rotator until all blocks could be collected.

3.1.2. Tissue Dissection
Individual tissue blocks were transferred to 35-mm dishes, placed on their
anterior face, and covered in ice-cold aCSF. The subventricular zone (approx
1.5 mm

W), including the ependyma and subependyma, was
dissected from the striatal side of the lateral ventricles ( Fig. 1 ) under a dissect-
ing microscope using surgical instruments (scalpel, dissecting scissors, and
needle probes).

× 0.2 mm, L ×

3.2. Single-Cell Dissociation
Single cells were dissociated enzymatically in stepwise fashion.

1. Dissected tissue was minced into fi ne strips with a scalpel.

2. Minced tissue was transferred and pooled in a single 15-mL polypropylene
tube.

3. Two milliliters of dissociation media was added and the tube was lightly vortex-
mixed at the lowest vortexer/mixer setting. Tubes were capped and incubated for
30 min at 37°C in an hybridization oven with rotation.

4. Five milliliters of enzyme inactivation media was added and the sample was
centrifuged for 5 min at 200 g .

5. The supernatant was removed. Ten milliliters of enzyme inactivation media
was added to the cell pellet and the suspension was transferred to a 50-mL
polypropylene tube. Tissue was dissociated by tituration through successive
10-mL, 5-mL, 1-mL, and pasteur pipets.

6. The suspension was transferred to a 30-mL syringe and forced through an
18-gauge needle into a 50-mL polypropylene tube.