Cd3 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).

Lymph node aspirates were collected into flow cytometry buffer and were analyzed immediately or stored at 4°C for a maximum of 24 hours. The flow buffer was composed of phosphate buffered saline (PBS) with 1% horse serum, 5mM ethylenediaminetetraacetic acid (EDTA) and 0.1% sodium azide. Flow cytometric immunophenotype of the neoplastic cells was determined using a FACScan instrument (BD Biosciences, Mississauga, Canada). Leukocytes were stained with a panel of 11 antibodies including: CD3_FITC (CA17.2A12, T lymphocytes), CD4_FITC (YKIX302.9, neutrophils and T-helper lymphocytes), CD5_FITC (YK1X322.3, T lymphocytes), CD8α_PE (YCATE55.9, cytotoxic T lymphocytes), CD14_PE (TUK4, monocytes), CD18_A647 (CA1.4E9, all leukocytes), CD21_PE (Ca2.1D6, B lymphocytes), CD22_PE (RFB4, Novus, B lymphocytes), CD34_PE (1H6, hematopoietic stem cells), CD45_FITC (YK1X716.13, all leukocytes) and MHCII (YK1X334.2, lymphocytes and monocytes) (Bio-Rad, Berkeley, CA, USA).