Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 103

Prepare a step gradient in an ultracentrifuge tube by adding 1 mL of
50 % sucrose solution (bottom) and 3 mL of 20 % sucrose solution
(top). Carefully add 9 mL (SW 40 Ti) or 8 mL (SW 41 Ti) of virus
stock solution onto the sucrose gradient. Ultracentrifugate at
160,000 g (30,000 rpm in SW 40 Ti or SW41 Ti rotor) for 90 min at
+4 °C. The virus will settle near the interface between the 20 % and
50 % sucrose layers and can be collected by discarding the medium
fraction and the bottom 0.8 mL consisting of 50 % sucrose.

3.11.1 Ultracentri-
fugation of Virus Stocks

Load virus onto the sample container of the Centriprep concentra-
tor as described by the manufacturer. Centrifugate the assembled
concentrator at an appropriate g -force (according to the manufac-
turer's recommendations) until the fl uid levels inside and outside
the fi ltrate collector equilibrate. Remove the device, snap off the
airtight seal cap, decant the fi ltrate, replace the cap, and centrifu-
gate the concentrator a second time. Decant the fi ltrate, loosen the
twist-lock cap and remove the fi ltrate collector. Collect the con-
centrated virus sample with a 1-mL disposable plastic pipette. If
further concentration of virus is desired, additional centrifugation
can be performed after decanting the fi ltrate.

3.11.2 Centriprep
Concentration

Use Matrex ® Cellufi ne™ Sulfate (Millipore) for virus concentra-
tion according to the manufacturer's recommendations. This
method allows effi cient removal of endotoxins and other contami-
nants. Follow the column packing procedures provided by the
manufacturer. Equilibrate column with adsorption buffer (0.01 M
phosphate, 0.1 M NaCl, pH 7.5) and load sample at pH 7.5. Wash
with several bed volumes of adsorption buffer to remove nonbinding
contaminants. Elute virus with elution buffer (1-2 M NaCl or KCl).

3.11.3 Affi nity
Chromatography
Concentration

3.12 Infection
of Cultured
Hippocampal Slices

Because neurons in organotypic hippocampal slices cultured
according to the roller-tube technique [ 23 ] are generally covered
by a layer of glial cells, viral particles do not easily penetrate into
the tissue. Moreover, the spread across the extracellular space of
brain tissue is limited. This problem has been solved by manually
injecting the infectious SFV particles into the extracellular space of
the tissue. For the microinjection of virus into slices, one can use
commercially available devices such as the Microinjector 5242
(Eppendorf). A cheaper and less complicated alternative is to insert
a micropipette containing the viral solution into an airtight
electrode holder (such as used for patch-clamp recordings in elec-
trophysiological experiments) and mount the holder on a micro-
manipulator (e.g., Narishige). Pressure can be applied with a 1-mL
syringe connected to the electrode holder via a plastic tubing for
injection of virus. The virus injection must obviously be performed
under sterile bio safety level 2 conditions. Slices are bathed in a
specifi c medium (cutting medium) to reduce excitotoxic injury
(i.e., spreading depression) during the virus injection procedure.