Anti cd11c pe n418

Single-cell suspensions of NALT cells were stained for 30 min at 4°C with appropriate combinations of fluorochrome-conjugated maternal antibodies in the presence of 0.2% bovine serum albumin (BSA) and fixed in 4% paraformaldehyde in PBS. Cells were stained for surface markers with anti-CD284 (TLR4)-PE-Cyanine7 (SA15-21, Biolegend) or anti-CD284 (TLR4)-PE (SA15-21, Biolegend), anti-CD11c-PE (N418, eBioscience), or anti-CD11c-PerCP-Cyanine5.5 (N418, eBioscience), anti-CD19-APC (6D5, Biolegend), anti-CD3-FITC (17A2, eBioscience), anti-CD11b-PE-Cyanine7 (M1/70, eBioscience), anti-Ly-6G/Ly-6C (Gr-1)-APC (RB6-8C5, Biolegend), anti-F4/80-PE (BM8, eBioscience), and anti-CD282 (TLR2)-PE (CB225, Biolegend) as needed. For intracellular staining, fixed cells were permeabilized and stained in saponin (0.1% in PBS; Sigma) with anti-CD289 (TLR9)-FITC (M9.D6, eBioscience) and anti-CD283 (TLR3)-PE (11F8, Biolegend) in the presence of 1.5% BSA. Samples were analyzed using a FACSCanto flow cytometer (BD Biosciences) and FlowJo software (Treestar, Ashland, OR).

C57BL/6 mice were infected by intranasal inoculation with 2.5 × 10⁶L. pneumophila 130b ΔflaA in 50 μL of PBS. Three days after infection, lungs were collected, minced and digested in 4 mL RPMI-1640 (Gibco) containing 3% FCS, 1 mg/mL DNaseI (Sigma-Aldrich) and 1 mg/mL Collagenase III (Worthington). Cells were collected by filtration through a 70-μM filter, centrifuged and red blood cells lysed by resuspension in buffer containing 150 mM ammonium chloride and 50 mM Tris-HCl (pH 7.5) for 5 min. After washing with PBS containing 0.1% BSA and 2 mM EDTA, cells were stained with anti-Ly6G-FITC (1A8, BD Pharmingen), anti-CD11c-PE (N418, eBioscience), anti-Siglec-F-BV421 (E50-2440, BD Horizon), anti-CD64-Alexa 647 (X54-5/7.1, BD Pharmingen) and anti-FcγII/III (2.4G2, WEHI monoclonal facility) for 30 min at 4 °C. Cells were washed and resuspended in FACS buffer with 0.25 μg/mL 7-AAD. Neutrophils were isolated using a Beckman Coulter MoFlo Astrios cell sorter into FCS and pellets of 10⁶ cells snap frozen at −80 °C. Purities were assessed post-sorting using the same gating strategy. Purified neutrophils were lysed on ice in PBS with 0.1% Triton X-100, and supernatants were cleared by centrifugation. Alternatively, lung tissues from the same mice were snap frozen at the time of harvest and processed as above.