Anti cd11c

Lungs from coinfected mice were collected on day 9 (2 d post-coinfection) and passed through a 70μM cell strainer. Cells were isolated by centrifugation through a 35% percoll solution and then stained with a 1:200 dilution of each antibody to determine which cell types express pro-IL-1β in the lung during coinfection. An initial surface stain was performed using anti-CD11b, anti-CD11c, anti-Gr1, anti-CD3ε, and anti-CD19 (TONBO biosciences, clones M1/70, N418, RB6-8C5, 145-2C11, 1D3). Then, cells were fixed with IC Fixation Buffer and permeabilized in 1x Permeabilization Buffer (eBioscience, 00-8222-49, 00-8333-56) followed by staining with anti-IL-1β pro-form (eBioscience, clone NJTEN3). Cells were distinguished based on the following gating strategies: CD3ε—CD19^- CD11b⁺ CD11c^- Gr1^- (macrophages), CD3ε—CD19^- CD11b⁺ CD11c⁺ Gr1^- (Dendritic cells), CD3ε^-CD19^-CD11b⁺CD11c^-Gr1⁺ (neutrophils and inflammatory monocytes), CD3ε⁺CD19^- (T cells), CD3ε ^-CD19⁺ (B cells), and lineage negative cells were considered mainly epithelial cells.

Cell surface staining were performed with the following antibodies: anti-CD3ε, and anti-CD4, anti-CD11b, anti-CD11c, anti-CD19, anti-B220, and anti-Ly-6G (TONBO Bioscience, San Diego, CA, USA); anti-IL-33R (ST2), anti-TCRγδ, anti-CD90.2, and anti-CD45 (BioLegend, San Diego, CA, USA); anti-TCRβ, anti-CD25, anti-CD44, anti-CD62L, anti-CD69, anti-CD103, anti-NK1.1, and anti-TER119 (BD Bioscience); and anti-CD127 (eBioscience, San Diego, CA, USA). The same procedures were conducted with the following antibodies for the intracellular staining: anti-IL-4 and anti-IL-5 (BD Bioscience); and anti-IL-13 (eBioscience). Fixable Viability Dye (eBioscience) was used to exclude dead cells. All samples were analyzed using the FACS Verse (BD Bioscience) and Flow Jo software programs (Tree Star Inc., San Carlos, CA, USA).

Heart inflammatory cells were isolated and processed as previously described [30 (link),31 (link)]. For the flow cytometric analysis of the surface markers and cytoplasmic cytokines, the cells were stained directly using fluorochrome-conjugated mouse-specific antibodies and analyzed using the FACSVerse instrument (BD Biosciences, San Jose, CA, USA). The following monoclonal antibodies were used for flow cytometric analysis: Anti-CD279 (PD-1, TONBO Biosciences, San Diego, CA, USA), anti-CD274 (PD-L1, TONBO Biosciences, San Diego, CA, USA), anti-CD273 (PD-L2, Invitrogen, Waltham, MA, USA), anti-CD45 (TONBO Biosciences, San Diego, CA, USA), anti-CD4 (TONBO Biosciences, San Diego, CA, USA), anti-CD8 (eBioscience, San Diego, CA, USA), anti-CD11b (BioLegend, San Diego, CA, USA), anti-CD11c (TONBO Biosciences, San Diego, CA, USA), and anti-Ly6G (BD Pharmingen, San Jose, CA, USA).