Polyplus jetprime reagent

For preparation of lentivirus, 1.3×10⁶ HEK-293FT cells were plated on a six-well plate precoated with 200 μg/mL poly-l-lysine and let to adhere overnight. pLVX plasmids containing H2B-GFP, H2B-tdTomato, MyrPalm-tdTomato (Zacharias et al., 2002 (link)), LifeAct-GFP, Wasabi or tdTomato under EF1 promoter together were mixed with psPAX2 (a gift from Didier Trono, Ecole Polytechnique Tédérale de Lausanne, Lausanne, Switzerland; Addgene plasmid 12260) and pCMV-VSV-G (a gift from Bob Weinberg, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Addgene plasmid 8454) at a molar ratio of 3:2:1, and cells were transfected using Polyplus jetPRIME reagent (Polyplus Transfection). After 24 hr, medium was replaced with complete DMEM supplemented with 0.075% sodium bicarbonate. Medium-containing viruses were collected after 24 hr and 48 hr.
For tumor cell infection, virus-containing media were mixed with 100 μg/mL polybrene (Sigma-Aldrich) and added to B16F10 cells (1 × 10⁶ cells on 35 mm tissue culture) for 30 min at 37 °C 5% CO₂. Cells were centrifuged for 30 min at 37 °C and 450 g. Then, 80% of the medium was replaced with complete DMEM supplemented with 0.075% sodium bicarbonate. After 3 days in culture, cells were sorted by FACSAriaIII. Cells were tested for mycoplasma, endotoxins, and bacterial contamination.

HEK-293FT (Invitrogen) were plated on 10 cm culture plates and co-transfected with a 2:1 molar ratio of pMIGII and PCL-Eco plasmids (both were provided by Dario A.A. Vignali, University of Pittsburgh) using Polyplus jetPRIME reagent (Polyplus Transfection). After 24 hr, the medium was replaced with complete DMEM supplemented with 0.075% sodium bicarbonate. Media-containing viruses were collected after 24 hours and 48 hr and centrifuged for 1 hr at 100,000 g. The pellet was resuspended gently in 1 mL media and let to recover overnight at 4 °C.
Prior to infection, splenic T cells were incubated on a plate precoated with anti-CD3 (0.5 μg/mL) in T cell medium containing high-dose IL-2 (1000 IU/mL). Next, 0.3 mL concentrated retroviruses were added to every group of 2×10⁶ cells with 10 μg/mL polybrene. Cells were incubated for 30 min at 37 °C in 5% CO₂ and centrifuged at 37 °C, 600 g, for 1 hr. Afterwards, 80% of medium was replaced and T cells were cultured for an additional 3 days in T cell media containing 1000 IU IL-2. Transduction efficacy was assessed by FACS as the percentages of GFP-expressing cells.