Profection mammalian transfection kit

The pseudotyped virus (PsV) neutralization assay was performed as previously described (11 (link)). Briefly, lentiviruses were packaged in HEK293T cells by transfection of 5 plasmids, 400 ng of HA, 100 ng of NA, 50 ng of TMPRSS2, 7 μg of CMVΔR8.2, and 7 μg of pHR′CMV-Luc with the Profection mammalian transfection kit (E1200; Promega). Viral particles were collected 48 and 72 h posttransfection and filtered through a 0.45-μm filter. Antisera at the indicated dilution were preincubated with a fixed amount of lentivirus and used to infect target 293A cells. Infection was quantified 72 h later with the Promega luciferase assay system (no. E1500). Fifty percent inhibitory concentrations were calculated with GraphPad Prism from neutralization curves. Preimmune sera were used as a negative control for each influenza strain tested in PsV assays.

Packaging of lentiviruses was performed by transient transfection of HEK cells. One day before transfection, HEK cells were seeded in a T75 flask at 1 × 10⁵ cells/cm² in DMEM supplemented with 10% FCS, 1 mM pyruvate, and 40 mg/ml gentamicin. The calcium phosphate precipitation method was used to co-transfect cells using 7.5 μg of gag/pol packaging plasmid psPAX2, 7.5 μg of pGIPZ-Foxp3-shRNA/Foxp3-cDNA transfer vector, and 4 μg of envelope plasmid pMD2.G using the Profection mammalian transfection kit (Promega, USA). Transfections were carried out in 10 ml of DMEM without antibiotics, and the cells were grown for 16 h. After that, the medium was replaced with complete DMEM, and after 48 h, the supernatant was collected. Centrifugation at 1,500 rpm for 5 min at 4°C was used to remove cell debris, which was then passed through a 0.45-μm pore PES filter. For concentration, 20 ml of supernatant was centrifuged at 4,000 rpm for 20 min at 4°C in a 12% polyethylene glycol (PEG-8000) solution. In serum and antibiotic-free DMEM, pelted viruses were re-suspended and the aliquots were kept at −80°C for further use (41 (link)).

1.25 μg of Env+ plasmids carrying rev-env sequences were co-transfected with 1.25 μg of pNL43 that carried a premature stop codon in the envelope gene and 0.625 μg of pHIvec2-GFP plasmid [78 (link)] into 293T cells using calcium phosphate (Profection mammalian transfection kit, Promega Inc.) [2 (link)]. The cell supernatant was changed 8-18 hrs post-transfection (4% FBS DMEM). Pseudovirions were harvested 48 h post-transfection, clarified by low-speed centrifugation, aliquoted into 0.5 ml portions, and snap-frozen in liquid nitrogen.

Lentiviral supernatants were generated by co-transfection of 293T cells with MA-1 as the expression vector and viral packaging plasmids psPAX2 and pMD2.G (Addgene ID 12260 and 12259 respectively). Cells were transfected using the calcium phosphate Profection Mammalian Transfection kit (Promega) according to the manufacturer’s instructions. The supernatant was collected twice: at 24 and 48 h following transfection and filtered through a 0.45 μm strainer. The supernatant was concentrated using Vivaspin (Sartorius) for the transduction of ER-Hoxb8 cells. For the transduction of fetal liver-derived HSPC, the supernatant was concentrated using ultracentrifuge (3:30 h, 20,000 rpm, 10 °C).
To transduce murine fetal liver-derived HSPC, we cultured the primary cells in 96-well plates with serum-free medium conditions as described. Each well was cultured with 50,000 cells. The viral titer was aimed to be approximately 50 MOI. Five µg/mL Polybrene was added to each well. We used spinfection for transduction at a rate of 1000 × g for 90 min at 22 °C. After centrifugation, the cells were incubated at 37 °C in 5% CO₂. Forty-eight hours post-transduction, the cells were collected, washed, and used for further applications. The transduction efficiency was measured using flow cytometry for GFP.