Pe conjugated anti dx5 and anti pe microbeads

DX5⁺CD3⁻ NK cells were first enriched from the naïve splenocytes of CD45.1⁺mice by positive selection with PE-conjugated anti-DX5 and anti-PE microbeads (Miltenyi Biotec) and then purified via flow cytometry sorting on a FACS DiVA with a purity >95%. 5 to 10 × 10⁵ NK cells were transferred by tail-vein intravenous injection into recipient CD45.2⁺ C57BL/6 or IL-18R^−/−mice. After two days, mice were infected by injection of 5 × 10⁶ pfu VV, i.p. or left naïve and sacrificed 24 hours post-infection for analysis.

NK cell cytotoxicity was performed by a standard 4-hour chromium-51 release assay as described [5 (link)]. Splenocytes were enriched for DX5⁺ NK cells by positive selection with PE-conjugated anti-DX5 and anti-PE microbeads (Miltenyi Biotec). YAC-1 target cells (ATCC), which are susceptible to NK cell-mediated cytotoxicity, were labeled with chromium-51 and then incubated with DX5⁺ cells at different effector:target ratios for 4 hours at 37°C. The specific release of chromium-51 into the supernatant, measured by counts per minute, assessed target cell lysis with no effector (spontaneous lysis) wells and 1% SDS (maximum lysis) wells used as controls. Specific release was calculated as (experimental – spontaneous)/(maximum-spontaneous) x100.