Biology Reference
In-Depth Information
13. ELISpot coating antibody solution: 4
μ
g/mL Anti-IFNg
AN18 diluted in PBS.
14. Detection antibody solution: 1
μ
g/mL Anti-IFNg R4-6A2-
biotin diluted in PBS 0.5 % FBS.
15. Streptavidin-Alkaline Phosphatase (ALP) solution (Sigma):
dilute 1:1,000 in PBS 0.5 % FBS.
16. BCIP/NBT solution (Sigma).
17. CFSE stock solution: Dissolve 5-(6-) carboxyfl uorescein diace-
tate succinimidyl ester (CFSE; Sigma ref. 21888) in 5 mM
dimethylsulfoxide. Store aliquots at −20 °C. The stock solu-
tion can be refrozen quickly after thawing. Prepare the diluted
CFSE just before use and do not freeze it.
3
Methods
1. In a 96-well dish prepare 1/10 serial dilutions of the virus
stock or sample using DMEM/5 % FBS in a fi nal volume of
100
3.1 Adenovirus
Titration by Anti-Ad
Staining (Infectious
Unit Titer)
μ
L. Add 100
μ
L of medium to all wells needed and then
L of concentrated virus to the fi rst column. Use three
lanes per sample (triplicate). Change tip every time after the
virus is taken to the next column and mixed. Prepare at least
ten serial dilutions, although this range can be adjusted consid-
ering an approximate titer of 10
8
infectious units (iu)/mL in a
cell extract and 10
10
iu/mL in a purifi ed virus. Do not add
virus to the last column (negative control) (
see
Note 1
).
2. Prepare a cell suspension of 10
6
cells/mL in DMEM + 5 % FBS
and add 50
add 10
μ
L to each well (50,000 cells/well). Select a proper
cell line according to the characteristics of the virus to analyze
(typically HEK293 cells).
3. Incubate the infected cells at 37 °C for 24-36 h (for HEK293
cells) (
see
Note 2
).
4. Carefully remove the medium with a tip connected to vacuum
pump or to a pipette. Avoid cells loss. Let the cells air-dry for
10 min.
5. Fix cells with 100
μ
L/well of 100 % methanol for 10 min at
−20 °C (this step can be extended for the desired time).
6. Prepare the primary antibody (anti-hexon, anti-fi ber, or anti-
adenovirus polyclonal) in PBS-Ca/Mg, 1 % BSA. For a hybrid-
oma supernatant (we use 2H×-2 hybridoma) prepare a 1/5
solution. For a purifi ed anti-adenovirus antibody prepare a
1/500 dilutions.
7. Remove the methanol from the titrating plate without contact-
ing the monolayer.
μ
