Ad293 cells

A recombinant E1, E3 deleted human adenovirus type 5 (AdHu5) vaccine vector encoding HIV-1 CN54gag (AdHu5-CN54gag) was used which has been described previously [13] (link). High titre virus stocks were prepared on AD293 cells (Cell Biolabs, Inc., San Diego, USA) and purified by the Native Antigen Company (Oxford, UK). Viruses were tested for endotoxin using the Kinetic-QCL assay (Lonza, Slough, UK), according to the manufacturer's instructions.

Mouse MIN6^{47 (link)} and βTC^{48 (link)} β-cells were maintained in DMEM containing 10% fetal bovine serum (FBS) (all from ThermoFisher Scientific, Waltham, MA) at 37 °C and 5% CO₂. Cells were split 1:4 at 70–90% confluence. PIs were formed by seeding 2–5 × 10⁴ cells/ml onto Petri dishes or 10⁵ cells/ml in 125-ml ProCulture spinner flasks^{29 (link)} (Corning, NY). In spinner flask PI cultures, half-volume medium changes were performed every 2–3 days. AD293 cells (Cell Biolabs Inc., San Diego, CA) used for recombinant adenovirus production were cultured in the same medium as above but supplemented with 1% MEM non-essential amino acids (ThermoFisher). Cell viability was determined by Trypan Blue staining (ThermoFisher) using a hemocytometer or a TC20 counter (Bio-Rad Laboratories, Hercules, CA).