Transduction of Cells with Lentiviral Vectors

Parental HT1080, HT1080_αHis, and HT1080_CD62L cells were seeded at 8x10³ cells per 96-well and incubated with serial dilutions of vector stocks. Transgene expression was analyzed 72 to 96 hours later by flow cytometry. Activated PBMC were seeded at 4x10⁴ or 8x10⁴ cells per 96-well, respectively, in TCM medium before CD62L-LV (5 µL or 10 µL) or VSV-LV (0.05 µL or 0.5 µL) were added. Where indicated, CD62L-LV transduction of PBMC was carried out in the presence of Vectofusin-1 (Miltenyi Biotec, Germany) as described previously (21 (link)). Cells were centrifuged at 850g and 32°C for 90 minutes, followed by the addition of TCM supplemented with cytokines. The medium was replenished every 2 to 3 days. Optionally, cells were passaged. Transgene expression was assessed by flow cytometry.