Biomedical Engineering Reference
In-Depth Information
PROTOCOL 12.7 Determining rAAV Infectious Particles by
Infectious Center Assay (ICA)/Fluorescent Cell Assay (FCA)
Equipment and reagents
•
C-12 cell line (ATCC)
•
DMEM (Cellgro)
•
FBS
•
Geneticin 50mg/ml (Invitrogen)
•
Gentamicin 50mg/ml (Invitrogen)
•
0.025% trypsin-EDTA
•
PBS
•
Purified rAAV virus (pTR-UF
3
as an example)
•
Adenovirus serotype 5 (Ad5)
•
T-75 flask
•
96-well plate
•
Sterile pipettes and tips
•
Sterile 50 ml tubes
•
Biological safety cabinet
•
CO
2
incubator
Fluorescence microscope (Nikon).
•
Method
Day 1: Seed C-12 Cell into a 96-Well Plate
1 Take a T-75 flask of C-12 cells; pour off old media.
v
2 Wash cells with 10ml of PBS and decant.
3 Add 2ml of 0.025% trypsin-EDTA to cells; incubate about 2 min.
4 Knock on the T-75 flask until the cells are detached completely.
5 Add 8ml of fresh media (DMEM/200
μ
g/ml of geneticin/200
μ
g/ml of gentamicin/5%
FBS) to the trypsinized cells; resuspend the cells by repeatedly pipetting up and down,
forming cell suspension.
6 Transfer 0.75ml of the cell suspension to 10 ml of fresh media in a 50 ml tube.
w
7 Add 100
μ
l per well of the cell suspension into a 96-well plate (or into appropriate
number of wells of a 96-well plate); incubate at 37
◦
C, 5% CO
2
overnight.
Day 2: Infect Cells
1 Make serial dilutions of purified rAAV virus (pTR-UF
3
as an example):
(a) Take a fresh 96-well plate and put 250
μ
l
x
of fresh media in the first well (A1).
(b) Put 225
μ
l of fresh media into the wells from A2 to A10.
