Biology Reference
In-Depth Information
analyzing a cell suspension at the microscope the viable cells
appear bright and white, while dead cells appear blue, due to
the presence of the dye in their cytoplasm. For samples analysis
and cell counting under the microscope it is used a hemocy-
tometer (Fuchs-Rosenthal or equivalent) that has two count-
ing chambers with engraved squares to facilitate cell counting.
The cell concentration is determined by Eq.
2
:
cells counted
numberofsquares
Cell concentrationcell mL
( /) =
×
dil
utionfactor
1
(2)
×
3
square volume cm
(
)
where the dilution factor corresponds to the dilution of the
sample in the dye and the square volume corresponds to square
area multiplied by the chamber depth; this volume depends on
the hemocytometer used.
4. The lysis solution is used to recover the intracellular viruses.
Tris buffer is known to stabilize adenovirus vectors and the
detergent Triton X-100 to cell lysis. Alternatively lyse cell with
four freeze-cycles. Viral samples prior titrations are clarified at
3,000 ×
g
for 10 min to remove debris that might interfere
with infection process.
5. Glass bioreactors with working volumes ranging from 1 to
15 L are available from several sources. As these glass bioreac-
tors are sterilized in autoclaves, the interior dimensions of the
equipment have to be considered. For higher bioreactor vol-
umes, most stirred tanks are made of stainless steal. In this
case, sterilization is carried in situ using clean steam.
6. We recommend the use of a well-characterized viral seed stock
if possible concentrated and purified either by standard ultra-
centrifugation procedures or chromatographic methods [
22
,
23
]. One important issue to take into consideration is the
storage conditions of the viral seed stock. Several factors, such
as pH and temperature, affect the stability of the adenoviral
vector preparations during storage [
24
,
25
]. Several formula-
tions have been developed for long-term storage in liquid and
lyophilized form [
26
-
28
] from several months to more than 1
year at 2-8 °C (for more information
see
refs.
29
-
32
).
7. The silanization ensures that the glass surface becomes less
hydrophilic, allowing carriage, storage, and experiments with
microcarriers. Not all microcarriers require silanization, so if
you are not using Cytodex™, verify the manufacture recom-
mendations. The washing solution for this procedure should
be prepared as follows: dissolve 56 g of potassium hydroxide
in 76 mL of distilled water with constant agitation in ice
