Symphony a3 analyzer

Tissues were minced and digested with 2.5  mg/ml collagenase D (Roche) for 30  min at 37°C. 100  μl of 100  mM EDTA was added to stop 1  ml of enzymatic digestion. Digested tissue was pipetted up and down 30 times using a glass Pasteur pipette and passed through a 70 μm nylon filter to acquire single-cell suspensions from lungs, spleen, lung draining-LNs, skin draining-LNs, and mesenteric draining-LNs. Cells were stained with the following monoclonal Abs: Phycoerythrin (PE)-conjugated to Siglec H and NK1.1; PerCP-Cy5.5-conjugated to B220; PE-Cy7-conjugated to CD11c and CD44; BUV395-conjugated to CD11b and CD4; fluorescein isothiocyanate (FITC)-conjugated to Ly6D, CD3, CD27, and CD62L; allophycocyanin-conjugated to CD19 and CD103; APC-Cy7-conjugated to CD45; and BV510-conjugated to Ly6C. The viability dye DAPI (#D9542, Sigma-Aldrich) was added immediately before each sample acquisition on a BD Symphony A3 analyzer (BD Biosciences). Data were analyzed using FlowJo (Tree Star, Ashland, OR). Antigen-specific antibodies and isotype controls were obtained from BioLegend, eBioscience, and BD Biosciences.

Single-cell splenocyte suspensions were prepared by mechanical dissociation of spleen tissue over 100-µm cell strainers (Corning) and subsequent ammonium-chloride potassium lysis (Gibco) to remove red blood cells. For the analysis of splenic myeloid cell populations, spleen tissue was minced and digested in 0.07 mg/ml LiberaseTM (729904; Roche) and 0.05 mg/ml DNAseI (D4513; Sigma-Aldrich) prior to 100-µm filtration and ACK lysis. Staining of dead cells and blocking of Fcγ receptors was performed by incubation with LIVE/DEAD Fixable Aqua Dead Cell Stain (L34957; Invitrogen) and anti-CD16/32 (clone 2.4G2) in plain PBS for 45 min on ice. Surface staining was performed in PBS with 2% calf serum, 2 mM EDTA, and 0.05% sodium azide for 30 min at 21°C. Intracellular staining of Tbet, FoxP3, and Bcl6 was performed using the eBioscience fixation and permeabilization kit (cat. 00-5523-00) according to the manufacturer’s instructions with a 20-h incubation on ice. Antibodies used for flow cytometry are listed in Table S1. Flow cytometry was performed using a BD Symphony A3 Analyzer, a BD LSR Fortessa Analyzer, or a BD LSR Fortessa X-20 Analyzer.

Lung-draining LNs were teased with 26G needles and digested in 1ml of 2.5 mg ml⁻¹ collagenase D (Roche) solution in 1X RPMI at 37°C for 30 min. After incubation, 100 μl of 100 mM EDTA was added to stop the enzymatic reaction. The cells were pipetted up and down with glass Pasteur pipets and then passed through a 70 μm nylon filter. Single cell suspensions were collected and centrifuged at 300 g for 5 min. Cells were stained with the following monoclonal Abs: Phycoerythrin (PE)-conjugated to Vα2, CD4 and CD64; PerCP-Cy5.5- conjugated to CD11b and CD4; PE-Cy7-conjugated to CD11c and CD44; BUV395-conjugated to CD11b; BUV805-conjugated to CD8a, fluorescein isothiocyanate (FITC)-conjugated to CD4, CD45.1 and CD103; allophycocyanin (APC)-conjugated to Vβ51, CD45.2, CD45 and CD103; APC-Cy7-conjugated to CD45.2 and MHCII; and BV510 conjugated to Ly6C and MHCII. The PE-conjugated MHC Tetramer H-2kb OVA SIINFEKL was purchased from MBL international. The viability dye 4’,6-diamidino-2-phenylindole (DAPI) (#D9542, Sigma) was added immediately before each sample acquisition on a BD Symphony A3 analyzer (BD Biosciences). Data were analyzed using FlowJo (Tree Star, Ashland, OR). Antigen-specific antibodies and isotype controls were obtained from BioLegend, eBioscience and BD Biosciences.