Cd107a 1d4b

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).

Cells were labeled for flow cytometry by incubation with the following fluorophore-conjugated antibodies: CD3 perCP-Cy5.5(17A2; Biolegend), Cd49b PE (DX5; Biolegend), NKp46 APC (29A1.4; Biolegend), CD11b FITC (M1/70; eBioscience), Ly6G PE (1A8; BD Pharmingen), G-CSF Receptor (S1390; Abcam), CD107a (1D4B; Biolegend). Flow cytometric analyses were carried out on a fluorescent -activated cell sorting (FACS) Fortessa (BD Biosciences) using BD FACSDiva Software.

Tissues were processed, single cell suspensions obtained, and cells were stained as described (Wherry et al., 2003). Cells were stained with LIVE/DEAD cell stain (Invitrogen) and with antibodies targeting KLRG1 (2F1/KLRG1, Biolegend), CD127 (A7R34, Biolegend), CD8 (53-6.7, Biolegend), CD44 (IM7, Biolegend), CD45.1 (A20, Biolegend), CD45.2 (104, Biolegend), CD62L (MEL-14, Biolegend), CD27 (LG.7F9, Biolegend), CD122 (TM-b1, eBioscience), CXCR3 (CXCR3-173, Biolegend), Bcl-2 (A19-3, BD PharMingen), Bcl-xL (H-5, Santa Cruz), Bcl-6 (K112-91, BD PharMingen), Eomes (Dan11mag, eBioscience), Ki-67 (16A8, BioLegend), Bim (C34C5, Cell Signaling Transduction), and MHC class I D^bgp^33–41 tetramer (NIH tetramer core). Intracellular cytokine staining was performed after 5 hrs ex vivo stimulation with gp^33–41 peptide in the presence of GolgiPlug (BD), GolgiStop (BD) and CD107a (1D4B, Biolegend). After stimulation, cells were stained with surface antibodies, following fixation with Fixation/Permeabilization Buffer (eBioscience) and then stained with intracellular antibodies for TNF (MP6-XT22, Biolegend), IFN-γ (XMG1.2, BD PharMingen), GrzmB (GRB17, Life Technologies) and MIP-1α (IC450P, R&D System) using Permeabilization Wash Buffer (eBioscience) according to manufacturer’s instructions. Cells were analyzed with LSRII (BD Biosciences) and FlowJo software (Treestar).