Live dead near ir stain

PBMCs from calves inoculated intranasally and intratracheally with 10⁴ p.f.u. BRSV, Snook strain, as described previously [37 (link)] were collected 6 days post infection. CD14⁺ and CD3⁺ cells were separated by Macs. as described above. and rested overnight in culture media at 37 °C. CD14⁺ cells were then infected with wt BRSV or rBRSVΔSH, or transfected with plasmids expressing SH or control plasmids. as described above. and incubated with UV-inactivated BRSV (m.o.i. of 3). 1×10⁵ CD14⁺ cells were plated into each well of a 96-well U-bottomed plate (BD Biosciences) and mixed with autologous CD3⁺ T cells at a ratio of 5 CD3⁺ to 1 CD14⁺ cell; Brefeldin A (10 µg ml⁻¹, Sigma) was added to each well. The plate was vortexed briefly and incubated for 4–6 h at 37 °C. Live T cells were stained with Near-IR Live/Dead Stain (Life Technologies) and mouse anti-bovine CD3 (clone MM1A, VMRD) conjugated to allophycocyanin (APC). Cells were fixed and permeabilized using BD Biosciences’ Cytoperm/Cytofix buffer following the manufacturer’s instructions. Intracellular IFN-γ was detected using mouse anti-bovine IFN-γ conjugated to phycoerythrin (PE; clone CC302, Serotec). A minimum of 50 000 live events were acquired in a BD LSRFortessa. Single/live events were analysed using FlowJo version 10.7.

All flow cytometry was performed on a Fortessa LSR II flow cytometer (Becton Dickinson) using DIVA software (Becton Dickinson) and analysed using Flow-jo (Treestar, Ashland, OR) software. Prior to surface staining cells were incubated with mouse Fc Block (Biolegend) for 5 minutes in the dark at 4°C, after which 50μL of the relevant antibody cocktail was added, and the cells were left to incubate in the dark at 4°C for 30 minutes. Surface antibodies were FITC – F4/80, APC- CD11b, PE-ABCA1 (Santa Cruz Biotechnology, Heidelberg, Germany) FITC-TF (Biorbyt, Cambridge, United Kingdom) or FITC-TF (American Diagnostica). After surface staining cells were resuspended in 200μl pre-diluted Near IR live/dead stain (Life Technologies) and left to incubate in the dark at 4°C for 15 minutes. For intracellular staining, cells were permeabilised with Foxp3 intracellular staining permeabilisation solution for 30 minutes (e-Bioscience). Intracellular staining was performed using directly conjugated antibodies (BV605-CD206 (Biolegend) and PE-Cy7-iNOS (e-Bioscience)) made up into a staining cocktail using permeabilisation buffer (e-Bioscience), 50μL of staining cocktail was added per well and staining took place at 21 °C in the dark.

Cell fractions were suspended in cold MACS-buffer containing PBS, 2 mM EDTA and 0.5% BSA. To reduce unspecific binding, FcR blocking reagent (Miltenyi Biotec) was added to all samples. Cells were stained using the mouse anti-human-CD133-phycoerythrin (PE; 293C2), -CD34-fluorescein isothiocyanate (FITC) and -CD271-allophycocyanine (all Miltenyi Biotec), -CD45-allophycocyanin-H7 as well as -CD45-Horizon-V500 (both BD Biosciences, Heidelberg, Germany) following incubation for 10 min in the dark at 4 °C. For optimal multicolor setting and correction of the spectral overlap single stained mouse isotype antibodies were considered as controls. The gating strategy was performed with matched isotype/fluorescence minus one control. After performing antibody staining 15 µM 4′,6-Diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, Waltham, MA, USA.) was added, cells were incubated for 2 min and then immediately acquired by BD™ LSRII flow cytometer and data were analyzed using FACS-Diva software, version 6.1.2 (both Becton Dickinson, Franklin Lakes, NJ, USA). Purity and viability of all cell isolations were analyzed using near-IR live dead stain (Thermo Fisher).

Splenocytes from NHPs were processed on ice immediately after killing. A total of 10⁶ cells per condition were left unstimulated or restimulated for 16 h with 10 μg/ml EDIII, 10⁷ PFUs DENV-2 16681, or 20 ng/ml PMA + 1ug/ml ionomycin as a positive control. Brefeldin A and monensin (Biolegend) were added at 1:2000 each for the last 5 h of stimulation. Cells were incubated with Near IR Live/Dead stain (Thermo), fixed with Cytoperm/Cytofix (BD), and then stained with CD3, CD4, CD8, IFN-γ, and TNF-α antibodies as described in Supplementary Table 6. Cells were acquired on the Attune NxT flow cytometer (Thermo) and results analyzed with FlowJo. The T-cell response was considered significant if the frequency of responding cells was greater than threefold that of the average of unstimulated cells.

C57BL/6 mice (Jackson) were infected with 10⁶ Uvitex 2B-labeled (10 µg/ml; 5 min) yeast cells administered intratracheally. Single cell suspensions were made from harvested lungs with a 70-µm cell strainer and treatment with collagenase D (1 mg/ml; Roche) and DNase (50 U/ml; Roche). Leukocytes were enriched by density sedimentation (60%/40% Percoll, 20 min, relative centrifugal force of 600) and collection of cells at the interface. Approximately 10⁶ cells were stained for cellular markers and fixed with 4% paraformaldehyde (30 min). The markers included CD64-fluorescein isothiocyanate, CD45-peridinin chlorophyll protein-Cy5.5, SiglecF-phycoerythrin-Cy7, Ly6C-BV650, CD11c-BV786, CD90.2-allophycocyanin (APC), B220-APC, MHCII-A700, Ly6G-BUV395 (all from BioLegend), and Near IR live/dead stain (Thermo, Fisher). Cells were analyzed with an LSRII flow cytometer (BD Biosciences), and data were processed with FlowJo software (version 10.1).

Lungs were perfused with 20 ml cold PBS, cut into small pieces and incubated with 1 mg ml^–1 collagenase (Thermo Fisher) and 50 U ml^–1 DNase I (Life Technologies) in PBS for 30 min at 37 °C. Samples were filtered through 70-μm nylon strainers, and red blood cells were lysed using 0.83% ammonium chloride before resuspension in FACS buffer (2% FCS and 0.05% sodium azide in PBS). Samples were stained for 30 min at room temperature with fluorescently labelled antibodies to CD45 (BioLegend, 30-F11), B220 (BioLegend, RA3-6B2), GL7 (BioLegend, GL7), CD95 (BioLegend, SA362F7), CXCR4 (BioLegend, L276F12), CD86 (BioLegend, GL-1), TCRβ (BioLegend, H57-597), CD4 (BioLegend, GK1.5), PD-1 (BioLegend, 29F.1A12) or CXCR5 (BioLegend, L138D7) or unlabelled anti-eMLV Env (83A25, in house), anti-mouse IgG (BioLegend, Poly4060), anti-mouse IgA (Southern Biotech, 11-44-2), anti-mouse IgM (BioLegend, RMM-1), anti-human IgG (BioLegend, M1310G05), anti-human IgA (Miltenyi Biotec, 130-114-002) or anti-human IgM (BioLegend, MHM-88), all at a 1:200 dilution in FACS buffer along with Near-IR Live/Dead stain (Thermo Fisher). Samples were run on an LSR Fortessa running BD FACSDiva v.8.0 or a Ze5 analyser running Bio-Rad Everest v.2.4 and analysed with FlowJo v.10. Gating strategies used for the identification of different cell types are shown in Extended Data Fig. 12a.

For digestion of total ear skin, mice ears were removed, cut into small pieces, and transferred into Hank’s medium (Ca²⁺ and Mg²⁺ free, Life Technology), supplemented with Liberase TM (0.15 mg/mL, Roche) and DNase I (0.12 mg/mL, Sigma-Aldrich) and incubated for 1h at 37°C. The cell suspension was filtered through a 70-µm cell strainer (Falcon) and rinsed with PBS supplemented with 5 mM EDTA (Life Technologies), 1% fetal calf serum and 0.02% NaN₃. Single-cell suspensions were stained with antibodies directed against CD45 (clone 104), CD11b (clone M1/70), Ly6C (clone HK1.4), and Ly6G (clone 1A8). LIVE/DEAD Near IR stain (Life Technologies) was used for exclusion of dead cells. All staining steps were carried out on ice. Cells were acquired on a Spectral Analyzer SP6800 (Sony), and the data were analyzed with FlowJo software (FlowJo LLC). The gating of the flow cytometric data was performed according to the guidelines for the use of flow cytometry and cell sorting in immunological studies (74 (link)), including pre-gating on viable and single cells for analysis. Absolute cell numbers were calculated based on a defined number of counting beads (BD Bioscience, Calibrite Beads), which were added to the samples before flow cytometric acquisition.