8. Scrape cells off flasks with a rubber policeman (not trypsin) at 7 to 10 days post-transfection and transfer to 50 ml conical tubes. Spin cells in a benchtop centrifuge and resuspend pellet in 2.0 ml HBSS or sterile PBS. Freeze cells in dry ice/methanol bath, and thaw in a 37oC water bath. Vortex vigorously. Repeat freeze/thaw/vortex for 3 more cycles (four cycles total). Do not let virus supernatants warm up. Spin samples briefly and store supernatant at -20oC.
9. Infect two 50% to 70% confleunt T-25 flasks of 911 or 293 cells using 30-50% of the above viral supernatants for each flask. CPE or cell lysis should become evident at 2 to 3 days post infection. Productive infections easily observed with the AdTrack vectors.
10. Collect viruses when a third to half of the cells are detached, usually 3 to 5 days post infection. Presence of the recombinant adenoviruses can be confirmed by Western blot and/or PCR (for PCR, take 5 µl virus sup plus 10 µl PCR-grade Protease K at 55℃ for 1 hour, then boil samples for 5 min. spin briefly, use 1 to 2 µl for PCR).
11. Scrape cells off and prepare viral supernatants as described in Step 8 above. You should have at least 107 infectious particles/ml at this stage, and often much more. Each round of amplification should give at least 10-fold more virus than present in the previous round.
12. To amplify further, repeat the infection of cells using 30-50% of the viral supernatant from Step 11, using T75 flasks instead of T25 flasks. Titers can be measured at any time, which is particularly easy with AD-Track vectors. Simply infect 293 cells with various dilutions of viral supernatant and see how many are green 18 hours later. Without AdTrack, viruses can be plaque titered or titered by limiting dilution using standard methods; we find these methods much less simple and quantitative than that employing the GFP marker, but these have to be used if GFP is not present.
V. Preparation of High Titer Viral Stocks
1. Plate 911 or 293 cells in T-75 flasks to be 90% confluent at time of infection (about 1x107 cells/T-75). Usually, fiften to twenty T-75 flasks are sufficient to make a high titer stock.
2. Infect cells with virus sup at a multiplicity of infection (MOI) of 5 to 10 PFU per cell. When all cells have rounded up and about half of the cells are detatched (usually at 3 to 4 days post infection), harvest and combine all flasks. Spin 5 min in a benchtop centrifuge (~500 g), and remove the supernatant.
3. Resupend pellet in 8.0 ml sterile PBS. Perform four cycles of freeze/thaw/vortex. Centrifuge lysate in Sorvall HS4 rotor at 6000 rpm (7000 g) 4oC for 5 min.
4. Weigh 4.4 grams of CsCl in a 50-ml conical tube, transfer 8.0 mls of clear virus sup to the tube, and mix well by vortexing. Transfer the CsCl solution (about 10 ml, density of 1.35 g/ml) to a 12 ml polyallomer tube for SW41 rotor. Overlay with 2 ml mineral oil. Prepare a balance tube. Spin the gradient in SW41 rotor at 32,000 rpm, 10oC, for 18 to 24 hours.
5. Collect virus fraction (about 0.5 to 1.0 ml) with a 3cc syringe and an 18g needle. Mix with equal volume 2X Storage Buffer (2X Storage Buffer = 10 mM Tris, pH 8.0, 100 mM NaCl, 0.1% BSA, and 50% glycerol, filter sterilized). Store virus stocks at -20oC.
6. Check viral titer by GFP (preferred) of by plaque assays (see below) or by immunohistochemical staining, or simply read OD at 260 nm. To read OD, add 15 µl virus to 15 µl blank solution (Blank Solution = 1.35g/ml CsCl mixed with equal vol 2X Storage Buffer) plus 100 µl TE/0.1% SDS; vortex 30 seconds, centrifuge 5 min. measure A260. One A260 unit contains ~1012 viral particles (particles:infectious particles =~20:1).
