Anti mouse cd3e fitc

The adipose tissues and bronchus of each group of lungs were removed. The lungs were then cut into pieces and digested in RPMI 1640 medium (VivaCell, Shanghai, China) containing 10% FBS (Sperikon Life Science & Biotechnology Co., Ltd., Chengdu, China) and collagenase VIII (250 U/mL; Sigma Aldrich, Saint Louis, MO, USA) at 37 °C for 30 min. Cell surface staining was performed by incubating the cells with the indicated antibodies on ice for 15 min. Cells were washed and analyzed on an LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) using FlowJo software (BD Biosciences, San Diego, CA, USA).
To measure the percentages of CD4⁺ and CD8⁺ cells among CD3⁺ mononuclear cells, the cells were incubated with anti-mouse CD3e-FITC (1:1200, eBioscience); anti-mouse CD4-APC (1:400, eBioscience); anti-mouse CD8a-PE (1:1600, BioLegend); anti-mouse CD45R-PerCP-Cyanine5.5 (1:800, eBioscience) for 20 min at room temperature.
To measure the percentages of macrophages and dendritic cells, cells were incubated with anti-mouse I-A/I-E-PE/Cyanine7 (1:3200, BioLegend); anti-mouse F4/80-FITC (1:100, eBioscience); anti-mouse CD11b-eFluor™ 450 (1:800, eBioscience); anti-mouse CD11c-PerCP-Cyanine5.5 (1:200, eBioscience); anti-mouse Ly-6C-APC (1:800, eBioscience) for 20 min at room temperature.

A total of 1 × 10⁶ cells in BALs from immunized and challenged mice were stained with extracellular (anti-mouse CD3e-FITC, CD4-APC, CD8-APC-Cy-7, CD69-PECy-5) (eBioscience, CA, United States) markers using established procedures (32 (link)). For intracellular cytokine staining, 2 × 10⁶ splenocytes were cultured for 4 days with medium only, peptide pools (1 μg/ml of each lipopeptide) or PPD (1 μg/ml). On day 5, brefeldin A (1.5 μg/ml, 1 X; eBioscience) was added, and cultured for 5 h at 37°C and subsequently stained for extracellular markers CD3-PE Cy7, CD4-PE Cy5, CD8-APC-Cy7, and intracellular cytokines IFN-γ-PE and IL-10-FITC using our previously reported procedures. For intracellular GrB staining, 2 x 10⁶ splenocytes obtained from mice were stained for extracellular markers CD3-PE Cy7, CD49b-Alexaflour 700, CD8-APC-Cy7, and intracellular GrB-Alexaflour 647, without ex vivo stimulation, using our previously reported procedures (31 (link)). Samples were run on LSR Fortessa SORP flow cytometer and analyzed using FACS-DIVA software (Becton Dickinson, Mountain View, CA, United States). Respective isotype-matched control antibodies were used to gate non-specific staining in each experiment. Gates were set to exclude 95% of isotype control antibody stained cells in all extracellular and intracellular staining experiments.