Fixable blue dead cell stain

PBMCs (1 × 10⁶) were stained with fixable blue dead cell stain (ThermoFisher, Waltham, Massachusetts and USA) or Zombie NIR™ Fixable Viability Kit (Biolegend, London, UK). This was followed by washes and surface marker staining with antibodies CD4-BUV395, CD8-AF700, CD19-AF488, and CD14-BV711 (Biolegend, London, UK) followed by subsequent washes. Following surface staining, to determine the expression of membrane lipid rafts cells were subsequently stained with a surrogate lipid raft marker CTB¹⁶, conjugated to FITC (1:100 dilution in PBS) (Sigma, Dorset, UK) followed by subsequent washes and fixation in 2% PFA before running on the flow cytometer. Altneratively, cells were fixed filipin complex (Sigma) as described previously [74 (link)]. Stains and washes were carried out in cell staining buffer (Biolegend). Appropriate unstained controls were used for lipid stains.
Data was acquired using a LSRFORTESSA X-20 (BD, Wiltshire, UK) flow cytometer and analysis performed using FlowJo Single Cell Analysis Software (TreeStar). Application settings were created and Cytometer Setup and Tracking (CS&T) (BD) beads were run to assess cytometer performance for each experiment.

Flow cytometry was performed on cryopreserved cells. As per the gating strategy in figure 1A, NK cells were identified using a combination of Fixable blue dead cell stain (Life technologies), CD3-Pacific Blue, CD56-PE-Cy7 and CD16- APC-Cy7 (BD Biosciences). NK cell receptor expression was assessed using combinations of CD158a-PerCP-Cy5.5 (eBioscience), CD158b-FITC, KIR3DL1-Alexafluor700 (both Biolegend), KIR2DL3-PE, KIR2DL1-APC (both R&D Systems), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). For intracellular staining, cells were fixed and permeabilized (PermA/B solution, Caltag) according to the manufacturers' instructions, prior to incubation with Perforin-PerCP-Cy5.5 (eBioscience). At least 1500 NK cells were acquired for all samples on either a five laser BD LSRFortessa or a four laser BD LSRII system, equipped with FACSDiva Version 8.8.3 (BD biosciences). Rainbow beads ensured a consistent, comparable level of fluorescence across all samples on different days of acquisition. Gates were set using fluorescence minus one or unstimulated samples where appropriate. The data were analysed using FlowJo version 9.5.3 (Treestar, OR, USA).

Cryopreserved peripheral blood mononuclear cells (PBMC) were stained with combinations of antibodies that comprehensively interrogate the breadth of NK-cell receptors including the killer immunoglobulin receptors (KIRs), the c-type lectin receptors (NKG2), and the natural cytotoxicity receptors (NCRs). These included CD158a-PerCP-Cy5.5 (eBioscience), KIR3DL1-Alexafluor700 (Biolegend), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC, CD158b-FITC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). Dead cells were excluded using the Fixable Blue Dead Cell Stain (Life Technologies) prior to surface staining and fixation. For perforin staining, samples were then permeabilized (Perm A/B, Caltag) and stained with anti-Perforin-PerCP-Cy5.5 (eBioscience). NK cells were identified as CD3 negative lymphocytes expressing CD56 and/or CD16. At least 1500 NK cells were acquired per sample. Fluorescence minus one was used to set gates. The data were analyzed with Flowjo v9.5.4 (Treestar).

Following euthanasia by CO₂ exposure, lungs were perfused with chilled phosphate buffered saline (PBS) and then mechanically dissociated using a GentleMACS dissociator (Miltenyi Biotec, NSW, Australia). Lungs and mediastinal lymph nodes were then incubated with 10 U/mL DNAse I and Collagenase IV (Sigma-Aldrich, NSW, Australia) for 20 min at 37 °C before dissociation through a 70-µm cell strainer, washing and resuspension in RPMI/10% FCS for antibody staining. Single cell suspensions were resuspended in FACS wash (2% FCS, 5 mM EDTA in PBS) and incubated for 30 min with a mixture of fluorochrome labelled monoclonal antibodies detailed in Supplementary Table 1, Fixable Blue Dead Cell stain (Life Technologies, Thermo Fisher Scientific, NSW, Australia), and anti-CD16/32 blocking antibody (clone 2.4G2). Samples were acquired on a BD LSR-II (BD) and analyzed using FlowJo™ analysis software (Treestar, USA).