Quicktiter adenovirus quantitation kit

HAdV-F41 (ATCC^® VR-930™) was grown in 50–60% confluent HEK-293 cells (ATCC^® CRL-1573™) in DMEM (ATCC^® 30-2002) supplemented with 1–2% FBS (ATCC^® 30-2020™). Infection was done with virus at passage five at an MOI = 1. After infection, when cells show clear cytopathic effect (round up with increased nucleus size), cultures were harvested with a cell scraper and transferred to falcon tubes. Cell suspensions were centrifuged at 700× g, 4 °C for 10 min, and cells were resuspended in culture medium discharging the supernatant. Samples were subjected to three freeze/thaw cycles (−80 °C and 37 °C), then centrifuged at 1500× g, 4 °C for 10 min. Supernatants were aliquoted in small volumes and kept at −80 °C until use. To determine viral titers, an aliquot of the virus preparation was used for titration in HEK-293 cells via immunohistochemistry using the QuickTiter™ Adenovirus Quantitation Kit (Cell Biolabs, Catalog no. VPK-109, San Diego, CA, USA), following instructions by the manufacturer. Two cell types were used for the in vitro infection models: human colorectal carcinoma HCT116 cells (ATCC^® CCL-247, Manassas, VI, USA). HCT116 cells were grown in McCoy’s 5A medium (ATCC^® 30-2007™, Manassas, VI, USA) supplemented with 10% FBS.

To prepare virus lysates, cells were harvested by centrifugation to avoid aspirate non-adherent cells, Ad-fosARE, WT300 and dl1520-infected 293 cells were subjected to three cycles of freezing and thawing. Viral titers infectious units (ifu/mL) were counted using the Adeno-X™ Rapid Titer kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer’s directions. Virus particles (vp)/mL were determined by a QuickTiter Adenovirus Quantitation kit (Cell Biolabs, San Diego, CA, USA). For in vivo experiments, viral extracts were purified using a Fast-Trap Adenovirus Purification and Concentration kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocols.