Brilliant violet 421 labeled anti cd11b

Inflammatory cells in lung tissue were analyzed to identify eosinophils (CD45+CD11b+Ly6C-SiglecF+), Th17 cells (CD45+CD4+IL-17A+), regulatory T (Treg) cells (CD45+CD4+CD25+Foxp3+) by flow cytometry as previously described (Zhao et al., 2013 (link); Cossarizza et al., 2017 (link); Dong et al., 2018 (link)). Briefly, the excised right lung was digested by using lung dissociation kits (MiltenyiBiotec Technology & Trading Co. Ltd, Shanghai, China) to prepare single cell suspensions following the instructions. And then the cells were stained with the monoclonal anti-murine fluorochrome-conjugated Abs and detected by an Attune NxT instrument (Life Technology). Besides, the antibodies used were bought from BioLegend, including eflour506-labeled anti-CD45, Zombie NIR™ Fixable Viability Kit, Brilliant Violet 421™-labeled anti-CD11b, APC-labeled anti-Siglec-F, PE-labeled anti-Ly-6C, APC-labeled anti-CD25, FITC-labeled anti-CD4, PE-labeled anti-IL-17. PE-labeled anti-Foxp3 was purchased from eBioscience.

To assess cytokine and biological marker expression on γδ T cells and CD11b⁺Gr-1⁺ cells, 2 × 10⁶ cells per 100 μl were incubated with the following fluorescence-labeled antibodies for 30 min at 4 °C; fluorescein isothiocyanate (FITC) labeled anti-CD3; phycoerythrin (PE)-Cy7-labeled anti-γδ TCR; allophycocyanin (APC)-labeled anti-Vγ1; PE-labeled anti-Vγ2; Brilliant Violet 421-labeled anti-CD11b; APC-labeled anti-Gr-1; and PE-labeled anti-IL17RA (BioLegend, San Diego, CA, USA). After surface marker staining, the cells were permeabilized with Fix/Perm buffer (BD Biosciences) for 15 min, then incubated with the following fluorescence-labeled antibodies: Brilliant Violet 421-labeled anti-IL17A; Brilliant Violet 421-labeled anti-interferon (IFN)-γ; and PE-labeled anti-transforming growth factor (TGF)-β. The staining panels were as follows: γδ T cell, FITC-labeled anti-CD3, PE-Cy7-labeled anti-γδ TCR, APC-labeled anti-Vγ1, PE-labeled anti-Vγ2, Brilliant Violet 421-labeled anti-IL17A/Brilliant Violet 421-labeled anti-IFN-γ; CD11b⁺Gr-1⁺ cells, Brilliant Violet 421-labeled anti-CD11b, APC-labeled anti-Gr-1, PE-labeled anti-TGF-β/PE-labeled anti-IL17RA.
All flow cytometry data were acquired on an LSRFortessa X-20 cell analyzer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).