Lentiviral Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 48

sucrose ( see Table 3 ) to create a sucrose cushion. Carefully
remove the pipette. Slowly add the remaining viral superna-
tant into the tube, being careful not to disrupt the cushion
( see Notes 8 and 9 ).
16. Carefully balance the tubes with PBS before placing them into
the rotor (should be within 0.1 g of each other) (Beckman
Coulter Optima L-80XP, SW32Ti rotor, Beckman Coulter,
High Wycombe, UK).
17. Continue with ultracentrifugation at 81,949 × g for 2 h at 4 °C.
18. Carefully remove supernatant and then drain the remaining
medium from the tubes by leaving the tubes on a paper towel
in an inverted position for approximately 15 min. Aspirate the
remaining droplets. It is essential to remove as much medium
as possible to prevent over dilution of the virus.
19. Add 60 l of TSSM formulation buffer (Table 4 ) to the bot-

tom of the tube and seal the tube with Parafi lm. Carefully place
the tube into a 50 ml conical tube and close the tube with the
lid. Allow the pellet to dissolve for 2 h or overnight at 4 °C.

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20. Next day proceed to resuspension of the virus pellet. Vortex
the ultracentrifugation tubes with the virus at low speed every
15 min for 15 s over 2 h. The tubes should be kept on ice
during the intervals.
21. Aliquot and store the lentiviral stock −80 °C.

In many in vitro applications, mainly vector entry and traffi cking
studies in neurons, visualization of the lentiviral particle is advanta-
geous and can provide a wealth of information regarding the spread
of virus upon infection (i.e., the endocytic traffi cking itinerary that
a vector follows). In order to label the particles with lipophilic dyes
(Vybrant DiO, Dil, DiD, DiR, Invitrogen, UK), an additional step
is required (Fig. 3 ). Labeling of the envelope with lipophilic dyes
with the method described below allows highly effi cient labeling of
the viral vectors without affecting their biological titer.

3.2.5 Labeling
Lentiviral Particles
with Lipophilic Dyes

1. Sixteen hours post-transfection replace media with 15 ml
Opti-MEM (Invitrogen, UK) containing 3.7 mM Vybrant dye
and incubated at 37 °C for 2 h.
2. Remove medium containing the Vybrant dye and proceed as
described in Sect. 3.2.4 (from Step 11 ).

For application of lentiviral vectors to target cells, it is essential to
accurately determine the transducing units of the lentiviral prepa-
ration. This will ensure that the vectors are viable and allow nor-
malization in variation between different preparations. Viral titers
can generally be affected by a number of factors, such as: (1) the
size of the gene of interest, as titers usually decrease with increasing

3.3 Titration
of Lentiviral Vectors