Adenovirus Purification Clontech

Test the Titer of the Adenoviral Stock

1. Determine the optimal amount of viral stock needed to infect 293 cells. To do this, test the viral titer in a small scale cytopathic effect assay, using culture conditions that closely approximate those that will be used for the actual adenoviral prep.

a. Seed low passage HEK 293 cells on a 12-well tissue culture dish at a density of 1x105 cells/cm2, using 0.28 ml/cm2 medium. At 1 ml/well, this is equivalent to ~3.57x10^5 cells/well.

b. Place the cells at 37oC and 5% CO2 while you dilute the adenovirus.

c. Infect the cells with a range of adenoviral concentrations. If the titer of adenovirus stock is known, aim for a multiplicity of infection (M.O.I.) of 1-2. if the titer is unknown, infect the cells with a range of dilutions, starting with 1 ml of stock and serially diluting 3X from there.

d. Incubate the cells at 37oC and 5% CO2 until the cytopathic effect is complete; this should take approximately 3-5 days.

Note: The cytopathic effect (CPE) refers to the morphological changes that the cells undergo after infection. Infected cells typically remain intact, but round up and may detach from the dish individually or in “grape-like” clusters that float in the medium. Optimally, the purification assay should be performed when 50% of the cells are detached, and the remainder are attached but rounded.

2. Use the results from the test titer to determine the optimal amount of virus to use in the actual purification prep. The full scale prep requires seeding several 150 mm cell culture dishes with 1.46x10^7 cells/dish. Therefore, if 0.1 ml of adenoviral stock optimally infected the 3.57x10^5 cells used to seed the test titer, 4 ml of virus is needed for each 150 mm dish in the actual prep.

Amplify Adenovirus in HEK 293 Cells

1. Detach low passage HEK 293 cells with trypsin, wash, and count.

2. In a 50 ml conical tube, dilute the cells to a final density of 1.83x10^6 cells/ml in a total of 40 ml.

3. Add virus to the cells and mix.

Note: Perform the test titer in Part A to determine the optimal amount of virus to add in this step.

4. Aliquot 33 ml of fresh medium onto each of five 150 mm cell culture dishes.

5 Aliquot 8 ml of virus-infected cells (from step 3) onto each of the five 150 mm cell culture dishes; make sure the cells are evenly distributed.
Note: This is equivalent to 1.46x10^7 cells/plate.

Incubate the cells at 37oC and 5% CO2 until the CPE is complete; this should take approximately 3-5 days.

Note: Optimally, the purification assay should be performed when 50% of the cells are detached, and the remainder are attached but rounded.

Harvest Adenovirus

1. Pellet the cells by centrifugation in a swinging-bucket centrifuge at 1,500 rpm (500 x g for Table-Top Beckman Centrifuge GH3.8 rotor) for 10 min.

2. Discard the supernatant.

3. Resuspend the pellet in 5 ml of fresh medium.

Note: Do not use PBS, as it inhibits some endonucleases.

Endonuclease is used below to remove contaminating cellular nucleic acids from filter-clarified cell lysate.

4. Lyse the cells with three consecutive freeze-thaw cycles; briefly freeze the cells in a dry ice/ethanol bath, then thaw them in a 37°C water bath.

5. After thawing for the final time, centrifuge the lysate at 3,000 rpm (~1,500 x g for Table-Top Beckman Centrifuge GH3.8 rotor) for 5 min to pellet the debris.

6. Collect and save the supernatant in a sterile centrifuge tube. Discard the pellet.

7. Add 5 ml Benzonase® Nuclease and incubate for 30 min at 37o C.

Note: Nuclease treatment decreases the viscosity of the solution so that it can be drawn more easily through the Adeno-X Purification Filter.

8. Measure the volume of the sample with a pipet, then add an equal volume of 1X Dilution Buffer (approximately 5 ml).

Note: The solution may turn slightly purple if you resuspended the original cell pellet in fresh medium (step 3).

9. Clarify the lysate by filtering it through the 0.45 mm Syringe-tip Pre-filter, using a 20 ml syringe (provided).

Purify Adenovirus

1. Equilibrate the Filter Assembly with 5 ml 1X Equilibration Buffer.

To do this, place the inlet tube into the buffer and draw the buffer into the syringe by pulling back on the plunger. Press lightly on the plunger to push the buffer through the Filter at a flow rate of
approximately 3 ml/min (this is approximately 1 drop/sec).

Note: Do not force residual air through the Filter. To remove air from the syringe, disconnect the Filter, force the air out of the syringe with the plunger, then reposition the Filter on the syringe.

2. Place the inlet tube into the clarified lysate, and draw the lysate into the syringe.

3. Load the adenovirus onto the Purification Filter. This is done by pushing the lysate through the Filter at a rate of ~3 ml/min to allow the virus to bind.

4. Transfer the inlet tube to a sterile tube containing 20 ml 1X Wash Buffer. Push the Wash Buffer through the filter at a rate of ~3 ml/min

5. Remove the Filter from the Assembly.

6. To elute the adenovirus, attach the Filter to a new 5 ml syringe containing 3 ml 1X Elution Buffer. Push 1 ml of Elution Buffer through the filter into a sterile 15 ml conical tube. Incubate the Filter at room temperature for 5 min, then push the remaining elution buffer through. Use residual air in the syringe to push any remaining virus through the Filter. Combine the eluate to obtain approximately 3 ml total.

7. Determine the adenoviral titer.

Notes:
• We recommend using the Adeno-X Rapid Titer Kit (Cat. No. 631028).
• For information on how to measure the adenoviral particle concentration by its optical density at 260nm (OD260 Assay), please go to www.clontech.com/support/titer

8. The adenovirus can be used immediately, or aliquoted and stored at –70°C.

For improved long-term stability, and proper tonicity for in vivo applications, we recommend a buffer exchange of the eluted adenovirus into 1X Formulation Buffer.

1X Formulation Buffer:
2.5% glycerol (w/v), 25 mM NaCl, and 20 mM Tris-HCl, pH 8.0
(GTS buffer; Hoganson, et al., 2002)