Adenovirus Infection Protocol in vitro and in vivo
a. Adenovirus Infection Protocol--Genemedi
For the reason that MOI varies in different cell lines, preliminary experiment is necessary to ensure a proper MOI of target cells before conducting formal experiments.
Note: MOI, multiplicity of infection, is the number of viral particles to infect one cell. An optimization test of MOI is strongly recommended as the real MOI to certain cells may be affected by methods of handling viruses in different labs.
1. Cell PreparationPlate robust target cells into 24-well plates at a density of 1 x 10^5/ml.
Note: The number of planted cells depends on the growth rate of a specific cell line. It should reach 50% to 70% confluence on the following day.
Prepare the virus in 10-fold dilution gradient, and ensure the MOI is within a range of 3 to 1000.
For adherent cells:
rAdVs containing target gene and same amount of control viruses should be added separately into two groups of cells and mixed well. The amounts of viruses to be used are based on size of container described in the following table. For MOI test in most cell types, a gradient of 3, 10, 30, 100, 300 to1000 at three replicates would be sufficient enough. Refresh medium in 4 to 8 hours. Protein of interests can be detected within 48-72 hours with fluorescence microscopy or WB, etc.
|Size of Container||Surface Area (cm2)||Volume of Medium||Volume of Viruses|
|96-well||0.3||100 µl||0.1-0.5 µl|
|24-well||2||500 µl||1-3 µl|
|12-well||4||1 ml||2-5 µl|
|6-well||10||2 ml||5-20 µl|
For example: If the tier of rAdV is 5 x 10^11 PFU/ml, dilute to 5 x 10^10 PFU/ml (10-fold) with complete growth medium of target cells. When there are 1 x 10^5 cells in one well, and the MOI is 1,000, required volume of diluted virus (5 x 10^10 PFU/ml) should be (cell number) x (MOI) ÷ (PFU/ml of rAdV) = 1 x 10^5 x 1000/5 x 10^10 (ml) = 2 µl. Thus, 2 ul of diluted virus should be added into this well.
For suspension cells:
Spin infection is a sufficient way to transduce suspension or semi-suspension cells. In brief, seal the cell culture plate by parafilm after adding viruses, spin in a low-speed swinging-bucket centrifuge at 200 g for 1 hour at 37℃, and culture cells at 37℃ overnight. Medium should be refreshed the next day.
If the condition is not allowed for spin infection, a centrifuge tube can be used instead by transferring cells into a tube and centrifuging at low-speed. Discard most of the supernatant after centrifugation, add viruses, and incubate at room temperature for 15-30 min. Then transfer the cell-virus mixture into a proper container, and culture at 37℃ overnight. Medium should be replaced the next day.
3. Determine Transduction Efficiency
48 to 72 hours post-transduction, fluorescent proteins can be observed when applicable, and the alteration of target gene can be analyzed at mRNA-level by qPCR or at protein-level by WB.
b. Animal experiment
Caution: Purification of rAdV is required for animal injection.
Take intravenous (i.v.) injection of mouse as an example:
Each mouse is injected with 5×109 – 1×1010 purified viral particles. If the titer is 1×1011 PFU/ml, 50-100 µl of purified rAdV will be required for each mouse. The amount of virus used for different experiments should be optimized accordingly.
Note: the volume of i.v. injection is usually 100 µl and can’t exceed 200 µl. If too much liquid is injected, the mouse is prone to congestive heart failure. Please consult our technical support for more information on other animal experiments using rAdV.
Detailed information of Adenovirus protocol can be seen in Adenovirus Protocol.