Biology Reference
In-Depth Information
(since it is an exothermic reaction); fill to 1,000 mL with
96 % ethanol and keep the solution at room temperature in
ambar-glass bottle for 14 day before use. All the solution
used in silanization protocol can be reused and should be
stored in an ambar-glass container.
8. FBS is used during inoculation and initial cell growth to pro-
mote adherence of cells to microcarriers.
9. Stirring rates herein presented are only valid for orbital shak-
ing motion with ∅ of 10 mm (IKA model HS 260 or
equivalent).
10. Use a thermomixer (37 °C, 300 rpm) to facilitate cell
detachment.
11. The virus titration can be performed by several methods.
Herein, a method for GFP expressing vectors is presented. For
viral vectors without fluorescent reporters, infectious particles
can be titrated using standard methods like TCID
50
or plaque
assay. The total particles can be determined by HPLC, quanti-
tative PCR, Nanosight or other, methods. More extensive
characterization of the virus obtained is normally performed
after purification.
12. One 175 cm
2
T-flask of 90 % confluence HEK 293 cells is suf-
ficient for three to four 24-well plates. Each plate is sufficient
to titrate two samples (six dilutions in duplicate).
13. One 175 cm
2
T-flask of 90 % confluence MDCK-E1 cells is
sufficient for 8 plates of 24 wells.
14. HEK 293 cells detach very easily from the culture surface
especially after infection. Careful is necessary when washing
cells; alternatively, this step can be removed for infected cells.
Collect also infected cells in suspension.
15. A supervisory control and data acquisition (SCADA) system
should be used for monitor and control process values during
preparation and run time, as exemplified in Table
1
.
16. pH calibration is performed before the electrode is installed in
the vessel using two commercial buffer of pH 4.01 and
pH 7.00. Calibration determines the “zero drift” and “slope”
of the electrode. First introduce the electrode inside the
pH 7 buffer. Connect the pH and temperature sensors to
the correct unit (where the bioreaction is going to run) to the
electrode. Start the data acquisition for pH and temperature.
When pH value is stable calibrate the “zero drift.” Change to
the pH 4 buffer and wait again for a stable pH value and cali-
brate the “slope.” Make sure that the “zero drift” and “slope”
values obtained are inside the limits of the electrodes used. If
not, start the calibration again or check if electrode is still
good for use.
