Biology Reference
In-Depth Information
10. Incubate at 37 °C with 5 % CO
2
for 24 h.
11. Discard media from two wells and wash cells carefully with
0.5 mL of D-PBS.
12.
For HEK293 cells
: Discard D-PBS and add 0.3 mL of Trypsin-
EDTA 0.05 %. Incubate cells at room temperature until detach-
ment is evident. Add 0.7 mL DMEM with 10 % FBS and
resuspend the cells.
For MDCK-E1 cells
: Discard D-PBS and add
0.5 mL of Trypsin-EDTA 0.25 %. When detachment becomes
evident add 0.5 mL DMEM 10 % FBS and resuspend the cells.
13. Transfer cells suspension to an eppendorf tube and determine
cell concentration (
see
Subheading
3.2.1
).
14. Dilute the viral suspension to be titrated on 3.5 mL tubes by
serial dilutions from 10
−1
to 10
−6
(0.3 mL of viral suspension
in 2.7 mL DMEM with 10 % FBS).
15. Discard media from each well of the cells.
16. Dispense 1 mL of the different viral dilutions onto the cells in
a way to have two replicates for each dilution.
17. For at least two wells add just culture medium (negative control).
18.
For HEK293 cells
: Incubate at 37 °C with 5 % CO
2
for 17-20 h
[
21
]. Transfer the content of each well to one 3.5 mL tube (
see
Note 14
). Add 0.3 mL of Trypsin-EDTA 0.05 % to each well
and incubate 30 s at room temperature. Add 0.7 mL DMEM
with 10 % FBS and resuspend the cells.
For MDCK-E1 cells
:
Incubate at 37 °C with 5 % CO
2
for 24 h. Collect cells from all
wells by detaching them as stated in
steps 11
-
13
.
19. Transfer cells suspension to the respective 3.5 mL tube to be
analyzed by flow cytometry and determine ip/mL according
to Eq.
1
(
see
Subheading
3.2.4
).
Flow cytometric data is acquired using a CyFlow
®
Space and data
analysis is performed using FlowMax
®
software from Partec. The
green fluorescence signal is collected by a photomultiplier tube
after passing through a 525 (±20) nm band pass filter (FL1).
3.2.3 Flow Cytometry
Analysis
1. Turn on the flow cytometer and make it ready for cell analysis.
2. Set the cytometric parameters to provide accurate discrimina-
tion between nonfluorescent negative cells and positive GFP-
fluorescence cells on a FL1 versus FSC density plot using
noninfected cells and highly infected cells as controls.
3. Start analyzing samples from the less to the higher infected.
4. Analyze GFP fluorescence of >1 × 10
4
single viable cells per
sample selected on FS versus SS scatter basis with high-
fluorescent cells gated.
5. A minimum of two dilutions showing 3-30 % GFP-positive
cells should be taken into account to calculate the viral titer.
