Cd86 bv421

Cells were collected and incubated in PBS plus 0.1% BSA (9998S; Cell Signalling Technologies) and 5 µl Fc block per million cells for 20 min. 50 µl of cells were then incubated with 50 µl antibody cocktail diluted in PBS and 0.1% BSA for 20 min on ice in the dark. Cells were washed in 2 ml PBS and fixed in 2% PFA (15710; Electron Microscopy Sciences) diluted in PBS prior to analysis. Cells were analyzed on an LSRII flow cytometer. Antibodies were purchased from BD Biosciences Antibody (CD14-Alexa488, 562689; CD119-PE, 558934; CD86-BV421, 562433; CD11b-bv421, 562632; CD163-FITC, 563697; CD169-PE, 565248; CD206-APC, 561763; CD16-Alexa647, 557710; Alexa488 isotype, 557703; Alexa647 isotype, 57714; PE isotype, 12-4015-82; BV421 isotype, 562438; CD16-Alexa647, 557710; Alexa488 isotype, 557703). Flow cytometry data was analyzed and plotted in FlowJo (BD Biosciences).

The polarization of macrophages was detected by flow cytometry after the preparation of the single-cell suspension from tumors. F4/80-BV605 (BD Biosciences, CA, USA), CD45-PE-Cy7 (BD Biosciences, CA, USA), and CD11b-AF-700 (BD Biosciences, CA, USA) were used together to identify the mononuclear immune cells in the tumor, then the proportion of CD86-BV421- (BD Biosciences, CA, USA) positive cells was identified as M1, and CD206-FITC- (Invitrogen, Carlsbad, USA) positive cells were identified as M2.

Cells were collected and incubated in PBS plus 0.1% BSA (9998S; Cell Signalling Technologies) and 5 µl Fc block per million cells for 20 min. 50 µl of cells were then incubated with 50 µl antibody cocktail diluted in PBS and 0.1% BSA for 20 min on ice in the dark. Cells were washed in 2 ml PBS and fixed in 2% PFA (15710; Electron Microscopy Sciences) diluted in PBS prior to analysis. Cells were analyzed on an LSRII flow cytometer. Antibodies were purchased from BD Biosciences Antibody (CD14-Alexa488, 562689; CD119-PE, 558934; CD86-BV421, 562433; CD11b-bv421, 562632; CD163-FITC, 563697; CD169-PE, 565248; CD206-APC, 561763; CD16-Alexa647, 557710; Alexa488 isotype, 557703; Alexa647 isotype, 57714; PE isotype, 12-4015-82; BV421 isotype, 562438; CD16-Alexa647, 557710; Alexa488 isotype, 557703). Flow cytometry data was analyzed and plotted in FlowJo (BD Biosciences).

Prior to antibody incubation, cells were stained with viability dye (Zombie™ dye, 1:500, Biolegend) for live cell/dead cell discrimination and incubated with Fc receptor blocking solution (Human TruStain FcX, BioLegend, CA) to prevent unspecific antibody binding. Extracellular antigens were stained using anti-human cluster of differentiation (CD)4-PE-Cy7, CD8-PE, CD14-PE-CF594 and CD19-FITC/PerCP-Cy5.5, CD20-APC-Cy7, CD25-BV605, CD27-PacificBlue, CD38-FITC, CD80-PE-Cy7, CD150-BV-421, major histocompatibility complex class II (MHC-II)-APC (all Biolegend, CA), CD19-PerCp-Cy5.5, CD40-PE-Dazzle, CD69-FITC, CD86-BV421, and CD95-PE (all BD Biosciences, NJ) antibodies. For analysis of intracellular cytokines, cells were permeabilized by adding fixation/permeabilization solution (Cytofix/Cytoperm, BD Biosciences, NJ) and stained with anti-human interleukin (IL)-6-FITC, IL-10-PE/CF594, and tumor necrosis factor (TNF)-Alexa Flour 700 (all BD Biosciences, NJ) antibodies. Apoptosis was evaluated using propidium iodide-PE and annexin V-FITC (both BioLegend, CA). Samples were analyzed using a LSRII Fortessa; FACS Diva (BD Biosciences) and FlowJo software were used to quantify flow cytometric data.

The expression of co-stimulatory molecules and maturation surface markers protein of iDCs, mDCs and mDCs co-cultured with primed WJ-MSCs were analysed by flow cytometer using the following antibodies: CD14- PE-CY7, CD83- PE-CY5, CD80-FITC, CD86- BV421, CD1a-PE, CD40-BV510, CD209-APC and HLA-DR- Percp-CY5.5, all antibodies are purchased from (BD Bioscience, USA). Cell proliferation and cell-surface marker expression of activated T cells (CD4+ and CD8+) were detected using the following antibodies: CD4-APC and CD8- PE-CY7 (Biolegend, USA). Activation of T cells was detected using CD69-PE (Biolegend, USA).