Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 102

Fig. 3 Metabolic labeling of SFV-transfected/infected BHK-21 cells. Cells electro-
porated with pSFV-NK1R and pSFV-Helper2 vectors ( lane 1 ) and infected with
SFV-NK1R virus stock ( lane 2 ) were metabolically labeled with 35 S-methionine
after 24 h and subjected to SDS-PAGE. p62, precursor p62 of SFV E2 and E3
membrane proteins; E1, SFV E1 membrane protein; C, SFV capsid protein; NK1R,
human neurokinin 1 receptor

buffer per 6-well plate and incubate 10 min on ice. Resuspend
thoroughly and load samples on 10-12 % SDS-PAGE and run gel
under standard conditions. Fix the gel in 10 % acetic acid, 30 %
methanol for 30 min at room temperature. Replace the fi xation
solution with Amplify ® (Amersham) and incubate 30 min at room
temperature. Dry the gel and expose on Hyperfi lm-MP for 2-24 h
(depending on signal) at room temperature or at −80 °C applying
radioactivity-intensifying screens. Develop fi lm to visualize radio-
active bands.

3.11 Infection
of Primary Neurons
in Culture

Primary neurons in culture are particularly sensitive to both physi-
cal and chemical exposure. Cell cultures of neurons should there-
fore be subjected to as little disturbance as possible. Minor
manipulations, such as removal and exchange of medium, can
already be harmful to the cultures. Add appropriate virus concen-
tration directly to primary neurons in culture and avoid any removal
or addition of medium. Perform a time-course study to obtain the
optimal expression pattern for the gene of interest. There are some
indications that the medium from BHK-21 cells (in which the
virus stocks are generated), under certain conditions, is toxic to
primary neurons in culture ( see Note 5 ). Different methods of
replacing the BHK-21 medium with a more appropriate and less
toxic medium for neurons have been developed.