Enhancing Viral Transduction in Natural Killer Cells Using a Cationic Polymer

Protocolo

1. Transduction of murine and human primary NK cells with lentivirus

1. Suspend mouse or human primary NK cells in a 24-well plate at 0.5 × 10⁵/mL of medium in the presence of green fluorescent protein (GFP) lentivirus supernatant at 5, 10, and 20 multiplicity of infection (MOI) and in the presence of Pb (8 µg/mL), PS (8 µg/mL) or dextran (8 µg/mL).
2. Centrifuge the plates at 1,000 × g for 60 min.
3. Without decanting the supernatant, culture the cells overnight (16-18 h) in a 37 °C incubator infused with 5.2% CO₂.
4. Wash with 10 mL of PBS and resuspend in 2 mL of complete Roswell Park Memorial Institute (RPMI) 1640 culture medium in the presence of interleukin 2 (IL-2, 300 U/mL).
5. Harvest the transduced NK cells on day 7 by gently tapping the flasks.
   1. Activate the harvested cells with titrated concentrations of plate-bound anti-natural killer group 2D (NKG2D) (A10) mAb by coating 96-well, highly protein-absorbent polystyrene plates with 2.5 µg/mL concentrations of anti-NKG2D mAb overnight.
   2. Wash each well with 100 µL of phosphate-buffered saline (PBS) three times before the addition of primary NK cells.
6. Collect culture supernatants using a multi-channel pipette between 16 and 18 h post-activation to quantify cytokines such as interferon-gamma (IFN-γ). Generate standard curves using the recombinant cytokines provided with enzyme-linked immunosorbent assay (ELISA) kits.
7. Test the NK cell viability using annexin-V/7-amino-actinomycin D (7-AAD) staining and determine the percent of necrotic cells among the transformed NK cells using a flow cytometer.
   1. To perform this assay, harvest the murine and human NK cells four days after transduction, wash two times with 10 mL of cold PBS at 500 × g for 5 min each, and incubate with an Annexin-V (PE)/7-AAD kit.
8. Use appropriate flow cytometry software to analyze the data. Select the live-cell population and analyze the expression of GFP (fluorescein isothiocyanate [FITC] channel) as a measure of viral transduction.

Materiais

Name	Company	Catalog Number	Comments
Dextran	Sigma-Aldrich	90-64-91-9
Polybrene (Pb)	Sigma-Aldrich	TR-1003
Protamine sulfate (PS)	Sigma-Aldrich	p3369
RPMI1640	Corning	10-040-CV
Interferon gamma (IFN-γ )	eBioscience	14-7311-85
Propidium lodide staining solution	BD	51-66211E
Lipofectamine 3000	Thermo Fisher	L3000015
Isoflurane	PHOENIX	NDC 57319-559-05
293T cells	ATCC	CRL-3216
T75 flasks	Corning	430641U
ELISA kits	Ebioscience	00-4201-56