Cd11c fitc clone hl3

The Abs used for cell-surface staining included: anti-CD3 Pacific Blue (clone 17A2), anti-CD8 PE Dazzle 594 (clone 53.6.7), anti-CD45 PE-Cy5 (clone 30-F11), anti-Ly-6G PE-Cy7 (clone 1A8), anti-Ly-6C APC-Cy7 (clone AL-21), anti-PD-L1 PE (clone 10F.9G2), anti-PD-1 APC (clone 29F-1A12), CD11b Alexa Fluor 700 (clone M1/70), anti-CD4 Brilliant Violet 570 (clone RM4-5), anti-CD44 PE-Cy7 (clone IM7), CD25 APC (clone 3C7) (Biolegend, San Diego, CA, USA), and CD11c FITC (clone HL3) (BD Biosciences, San José, CA, USA). The abs for intracellular staining were anti-FoxP3 Alexa Fluor 488 (clone MF23) (BD Biosciences) and anti-CTLA-4 PE (clone UC10-4F10-11) (Biolegend). A LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, Thermo Scientific, Eugene, OR, USA) was used for dead cell exclusion. The abs used for intracellular cytokines evaluation were anti-IFNγ Alexa Fluor 700 (clone XMG1.2), TNFα PE-Cy7 (clone MP6-XT22), IL-2 FITC (clone JES6-5H4) (BD Biosciences), anti-perforin (clone S16009A), and anti-granzyme B (QA16A02) (Biolegend).

The following Abs were used for cell surface staining: anti-CD3 Pacific Blue (clone 17A2), anti-CD8 PE Dazzle (clone 53.6.7), anti-CD45 PE-Cy5 (clone 30-F11), anti-Ly-6G PE-Cy7 (clone 1A8), anti-Ly-6C APC-Cy7 (clone AL-21), anti-PD-L1 PE (clone 10F.9G2), anti-PD-1 APC (clone 29F-1A12), CD11b Alexa Fluor 700 (clone M1/70), B220 PE (clone RA-3-6B2) (Biolegend, San Diego, CA, USA), anti-CD45 PE (clone 30-F11), anti-CD4 PerCP (clone RM4-5), Gr1 PE-Cy7 (clone RB6-8C5), CD11c FITC (clone HL3), and anti-CD44 APC (clone IM7) (BD Biosciences, San José, CA, USA). A LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, Thermo Scientific, Eugene, OR, USA) was used for dead cell exclusion.

Four days after challenge with RSV A2, mice were sacrificed, and adult and neonate tracheas were cannulated and washed with 700 and 300 μL of PBS, respectively. After BAL fluid was centrifuged, supernatant was stored at -80°C until analysis. Cells were incubated with violet fluorescent live-dead discriminator (Invitrogen, Eugene, OR) for 10 min at room temperature and then washed with 1 mL of PBS and blocked for 5 min with purified CD16/CD32 Fc (clone 2.4G2; BD Pharmingen, San Jose, CA). After blocking, 50 μL of antibody cocktail containing anti-CD45-APC (clone 30-F11), CD11c-FITC (clone HL3), Ly-6G (Gr-1)-PE-Cy7 (clone: 1A8), and Siglec F-PE (clone E50-2440; all from BD Pharmingen) were added to cells and incubated at 4°C for 30 min. Cells were subsequently washed two times with PBS (2% FBS) and fixed with 200 μL of paraformaldehyde. Cells were analyzed by using a BD FACS LSR II flow cytometer and data were analyzed with FlowJo software (version 10; Tree Star, Ashland, OR). The cytokines in BAL supernatant was measured using the mouse Th1/Th2/Th17 BD Cytometric Bead Assay Kit (BD Biosciences, San Jose, CA) according to the manufacturer’s recommendations. The cytokine levels were also analyzed using the Mouse Magnetic Luminex Screening Assay (R&D, Minneapolis, MN) [26 (link)].