Ly49h 3d10

Cell-surface staining was performed with fluorophore-conjugated antibodies against the following proteins: NK1.1 (PK136, Tonbo), CD11b (M1/70, Tonbo), CD27 (LG.3A10, BioLegend), KLRG1 (2F1, eBioscience), CD69 (H1.2F3, BioLegend), Ly49H (3D10, eBioscience), CD107a (1D4B, BioLegend), CD45.1 (A20, BioLegend), CD45.2 (104, Biolegend), TCRβ (H57–597, BioLegend), IFN-γ (XMG1.2, BioLegend), and Ly49D (4E5, BioLegend). Unless otherwise indicated, NK cells were defined as TCRβ-NK1.1⁺ cells. Intracellular cytokine staining was performed with the Cytofix/Cytoperm Plus Kit (BD). NK cells were enriched from spleens as mentioned above, stained with cell-surface antibodies, and then incubated with various dyes in Hank’s balanced salt solution plus Mg and Ca as follows: 100 nM Mitotracker Green (Life Technologies) for 30 min at 37°C to measure mitochondrial mass, 100 nM TMRE for 30 min at 37°C to measure mitochondrial membrane potential, 5 μm MitoSOX red (Invitrogen) for 15 min at 37°C to measure mitochondria-associated ROS, or 1:400 Cyto-ID autophagy detection reagent (Enzo Life Sciences) for 30 min at 37°C to measure autophagosomes. Flow cytometry and cell sorting were performed on the LSR II and Aria II cytometers (BD Biosciences), respectively. For experiments involving real-time PCR, cell populations were sorted to >95% purity. Data were analyzed with FlowJo software (Tree Star).

Fc receptors were blocked with anti-CD16+CD32 mAb (2.4G2) before staining. To monitor NK cells in peripheral blood weekly after MCMV infection, 50 μL of heparinized blood was lysed by ACK buffer and stained with anti-TCRβ (H57-597; BioLegend), -NK1.1 (PK136; BioLegend), -Ly49H (3D10; eBioscience), -CD45.1 (A20; BioLegend), -CD45.2 (104; Tonbo Biosciences) and -KLRG1 (2F1; BioLegend). Cells were analyzed on a BD LSR II flow cytometer using FlowJo software (Tree Star).