Apc labeled anti cd86

Caco-2 cells treated with FnEVs or TNF-α or inhibitors were harvested for apoptosis assay. The cells were washed with cold PBS to dissociate into single cells and then were treated with PI and FITC Annexin V (BD Pharmingen FITC Annexin V Apoptosis Detection Kit I). After 15 min of staining, the cells were assayed by flow cytometry, and the data were analyzed using the FlowJo software version 8 (FlowJo, Ashland, OR, United States). For phenotypic identification of macrophages, cells were stained with FITC-labeled anti-CD68 mAb (BD Pharmingen, USA) versus APC-labeled anti-CD86 (BD Pharmingen, USA) or FITC-labeled anti-F4/80 (BD Pharmingen, USA) mAb versus APC-labeled anti-CD86 (BD Pharmingen, USA), respectively. The percentage of CD86+ macropahges was immediately examined by flow cytometry.

DCs cultured with or without aspirin were prepared for surface marker expression analysis by FACS staining. Cells were incubated on ice for 15–30 min with specific monoclonal antibodies (mAbs): FITC-labeled anti-CD11c, PerCP/Cy5.5-labeled anti-MHC class II, PE-labeled anti-CD80, and APC-labeled anti-CD86 (BD Biosciences, USA). After staining, the labeled cells were analyzed with a BD FACSCanto II flow cytometer (BD Biosciences).