Cd206 fitc

To evaluate Treg polarization, PBMC-derived lymphocytes cocultured with cASC-EVs were harvested. Obtained cells (1 × 10⁶⁾ were suspended in 100μL DPBS and 1 μL of each primary antibody against the following proteins: FOXP3-PE (eBioscience, San Diego, CA, USA; 1:100), CD3-FITC (MCA1774F; Bio-Rad, San Diego, CA, USA; 1:100), CD206-FITC (eBioscience, San Diego, CA, USA; 1:100) and CD11c-PE (eBioscience, San Diego, CA, USA; 1:100). After incubation for 1 h at 23 ± 2°C, the cells were washed with DPBS. Unstained cells were used as controls for autofluorescence. Cell fluorescence was analyzed with a flow cytometer (FACS Aria Ⅱ; BD bioscience). The results were analyzed using FlowJo 7.6.5 software (Tree Star, Inc., Ashland, OR, USA).

The following directly conjugated anti-human monoclonal antibodies were used: CD4-FITC, CD4-APC, CD8-PE, CD163-PE, CD206-FITC, CD86-PE-Cy5, CD64-APC, CD273-PE, TLR-4-PE-Cy7, CD40-FITC, HLA-DR-PECy5 and CD274-APC (eBioscience, San Diego). Saturating amounts of antibody were used to stain approximately 3 x 10⁵ cells in staining buffer (1 x PBS, 2% FBS) at a final volume of 20 μl for 30 min at 4°C protected from the light. All samples were washed with staining buffer and resuspended in 200 μl of FACS Buffer. Samples were examined in a FACSCalibur (BD Biosciences) and analysis was performed using FlowJo software (Tree Star, Inc., OR).

Liver leukocytes of infected mice were prepared by Ficoll-Hypaque (Hao Yang Biological Manufacture, Tianjin, China) gradient centrifugation after being dispersed through a 70 µm nylon strainer. For flow cytometry analysis, peritoneal macrophages and liver leukocytes were washed twice and suspended in PBS containing 0.1% BSA and 0.05% sodium azide. A total of 1 × 10⁶ cells per 100 µl were incubated with F4/80-PE-Cyanine 5 (eBioscience, CA, USA), CD206-FITC (eBioscience, CA, USA) and CD16/32-PE (eBioscience) for 30 min at 4 °C in the dark. After washing twice, the cells were pelleted and resuspended. Data were acquired on a FACScan flow cytometer (Beckman Coulter) and analysed using FlowJo software (Treestar, San Carlos, CA, USA).

Stained cells underwent analysis utilizing FACS Canto II, and the resultant data were processed utilizing FlowJo version 10 software (Ashland, OR, USA). The following antibodies procured from eBioscience (Thermo Fisher Scientific, Waltham, MA, USA) were employed for the delineation of innate lymphoid cells: Biotin-CD3e (100304; clone: 145-2C11; 1/200), Biotin-CD45R/B220 (103204; clone: RA3–6B2; 1/200), Biotin-Gr-1 (108404; clone: RB6-8C5; 1/200), Biotin-CD11c (117304; clone: N418; 1/200), Biotin-CD11b (101204; clone: M1/70; 1/200), Biotin-Ter119 (116204; clone: TER-119; 1/200), Biotin-FceRIa (134304; clone: MAR-1; 1/200), FITC-Streptavidin (405202; 1/500), PE-Cy7-CD127 (135014; clone: A7R34; 1/100), Pacific Blue-CD45 (103116; clone: 30-F11; 1/100), PE-GATA-3 (clone: TWAJ; 1/50), APC-RORγ (clone: AFKJS-9; 1/50), and Fixable Viability Dye eFluor 780 (1/400) [20 (link),21 (link)]. Additionally, the following antibodies (eBioscience, San Diego, CA, USA) were utilized for the discrimination of M1 and M2 macrophages: APC-CD45.2 (17045482; clone: 104; 1/50), PE-F4/80 (12480182; clone: BM8; 1/50), APC-Cy7-CD11b (47011282; clone: M1/70; 1/50), FITC-CD206 (MA516870; clone: MR5D3; 1/50), and PE-Cy7-CD11c (25011482; clone: N418; 1/50) [22 (link)].