Biology Reference
In-Depth Information
8. Wash each well twice with 100
L PBS-Ca/Mg, 1 % BSA (ions
prevent cells detachment but PBS without ions can be also
used). During washing steps it is very important avoid air-dry
the cells.
9. Remove washing solution and add 50
μ
μ
L of the diluted Ab to
each well.
10. Incubate for 1-2 h at 37 °C.
11. Wash three times each well with 100
μ
L of PBS-Ca/Mg 1 %
BSA.
12. Dilute the secondary antibody (FITC- or Alexa488-
conjugated) in PBS-Ca/Mg, 1 % BSA. For most commercial
antibodies dilution 1/300 is recommended. Remove washing
solution and add 50
L of the dilution to each well and incu-
bate 1-2 h at 37 °C in dark.
13. Wash three times each well with 100
μ
L of PBS-Ca/Mg, 1 %
BSA. Try to avoid long exposition to light.
14. In an inverted fl uorescence microscope quantify the number of
green cells in each well (
see
Note 3
). Concentration will the
mean of the triplicate multiplied by the dilution factor. Note
that 100
μ
L of each dilution were used so a tenfold factor must
be applied to obtain the iu/mL titer.
μ
1. Tripsinize and prepare a 3 × 10
5
cells/mL suspension in DMEM
5 % FBS of HEK293 or the cell line tested for permissiveness.
2. Prepare serial dilutions of the virus. For cells that are easily
infected use 1/5 dilutions. For cells that need high MOI to be
infected use 1/2 or 1/3 serial dilutions. Prepare 11 serial dilu-
tions with DMEM/5 % FBS of each virus in a 96-well plate
with a fi nal volume of 50
3.2 Ad Bioactivity
Titration Assay (IC
50
)
L/well. Change pipette tips every
time after virus is taken and mixed in the next well.
3. In the last column (column 12) add a volume of 50
μ
L/well of
DMEM/5 % FBS but no virus. This will be the negative
control.
4. Decide the highest vp/cell or iu/cell (MOI) desired to infect
the cells in fi rst column in order to obtain complete cytopathic
effect (CPE) in 6-10 days. For example 5333.3 vp/cell or 100-
300 iu/cell. The vp/mL needed to infect the fi rst column is
5333.3 vp/cell × 30,000 cell/well = 1.5 × 10
8
vp/50
μ
μ
L =
L.
5. Dilute the viruses in order to obtain 3 × 10
6
vp/
3 × 10
6
vp/
μ
μ
L. Prepare a serial
dilution by transferring either 12.5
μ
L (1/5 dilution) or 25
μ
L
(1/3 dilution) to 50
L of DMEM 5 % FBS in the next column of
wells. Leave the last column without virus (negative control).
μ
6. Add 100
L of cell suspension to each well, from the negative
control column to the most concentrated.
μ
