Biology Reference
In-Depth Information
3.6 Rough Titer
Estimation
HC-Ad vector preparations possess a physical particle titer (i.e., the
concentration of physical HC-Ad vector particles per volume), an
infectious titer (i.e., the number of infectious vector particles able
to deliver their genomes into cells per volume), and a contamination
with helper virus. All three parameters should be determined to
fully characterize HC-Ad vector preparations and a wide variety of
DNA-based methods like qPCR and slot-blotting are available.
For the generation of high-titer HC-Ad vector stocks from purifi ed
HC-Ad vector it is suffi cient to roughly determine the physical
particle titer by OD260 ( see Note 20 ).
1. Add 79
L aliquot of
purifi ed HC-Ad vector as obtained in step 18 of the purifi ca-
tion protocol.
2. Add 1
μ
L of 50 mM HEPES pH 8.0 to the 20
μ
L of a 10 % solution of SDS in water (w/v).
3. Mix and incubate at 56 °C for 10 min.
4. Allow the sample to cool down to room temperature. Do not
put the sample on ice.
5. Measure OD at 260 nm.
6. Calculate the physical particle titer: OD260 × 5 × 1.1E12 = vp/mL
μ
3.7 Large-Scale
Preparation of HC-Ad
Vectors
Large-scale preparations of HC-Ad vectors are most conveniently
done with 116 cells in spinner culture. While 116 cells in spinner
culture can be infected with crude cell lysates obtained from serial
vector amplifi cations it is recommended to use purifi ed medium-
scale preparations obtained by the protocol described in
Subheading 3.4 . The reason for that is that it is diffi cult to evaluate
the CPE of cells in spinner culture and using defi ned amounts of a
purifi ed and titrated medium-scale vector preparation helps to
circumvent this pitfall.
1. Allow ten 150-mm dishes of adherent 116 cells to grow to
90 % confl uency.
2. Aspirate the medium from the cells and add 3 mL of pre-
warmed trypsin per dish.
3. Trypsinize cells for 1 min at room temperature and aspirate
trypsin.
4. Resuspend the cells of the ten dishes in 300 mL of fresh, pre-
warmed, MEM-Joklik supplemented with 5 % FCS, 0.1 mg/
mL hygromycin, 100 U/mL penicillin-streptomycin, and
2 mM glutamine. Transfer the cell suspension to a 3 L spinner
fl ask.
5. Incubate at 37 °C and stir cells at 60 rpm.
6. On the next day take an aliquot of the suspension, microscopi-
cally examine and count the cells. Add 0.5 L of fresh medium
supplemented as described in step 4 .
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