Dx 5 microbeads

For the in vivo stimulation and preparation of NK cells, 150 µg of Poly I:C was intraperitoneally injected. At 16 h after injection, mice were sacrificed and splenic NK cells were isolated using DX-5 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).
For in vitro stimulation of NK cells, Naïve splenic NK cells were isolated using DX-5 microbeads. 1 × 10⁶/mL of sorted cells were cultured with 50 µg/mL of Poly I:C and 200 U/mL of IL-2 (PeproTech, Cranbury, NJ, USA) for 16 h.
For the generation of IL-2 activated NK cells (also known as Lymphokine Activated Killer cells or LAK cells), 4 × 10⁶ cells/mL of whole splenocytes were cultured in a 100 mm dish with 1000 U/mL of IL-2 for 72 h. Plate unbounded cells were removed and the remaining cells on the plate were further incubated with a 1:1 mixture of fresh medium containing 500 U/mL of IL-2 and conditioned medium for four days. These cells were composed of over 80% NK cells (NK1.1⁺TCRβ⁻ cells).

NK cells were sorted from spleens of control and LDV infected mice using DX5 Microbeads (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. 7-AAD/CFSE Cell- Mediated Cytotoxicity Assay Kit (Abcam, Cambridge, UK) was used to assess by flow cytometry NK cell cytotoxicity against AB1 tumor cells and Yac-1 mouse lymphoma cells. Background of target cells without effector cells was subtracted.