Cd54 pe

Murine monocytes were isolated from the bone marrow of C57BL/6 mice by magnetic depletion (EasySep™ Mouse Monocyte Isolation Kit, STEMCELL Technologies Inc., Vancouver, BC, Canada). 5 × 10⁴ cells were cultured in 48-well plates in sEV-free RPMI medium and treated for 8 h with 5 µg of the respective sEV fractions referred above, as determined by BCA assay. Changes in PD-L1, HLA-DR and ICAM-1 expression were evaluated by flow cytometry (BD LSR Fortessa, BD Biosciences, San Jose, CA, USA). The following antibodies were used: PD-L1-PerCP (Biolegend, San Diego, CA, USA, #46-5982-82), HLA-DR-AlexaFluor700 (eBiosciences, San Diego, CA, USA, #56-5321-82), CD54-PE (Biolegend, San Diego, CA, USA, #116108), CX3CR1-BV711 (Biolegend, San Diego, CA, USA, #149031), Ly6C-APC-Cy7 (Biolegend, San Diego, CA, USA, #128015), CD11b-PeCy7 (Biolegend, San Diego, CA, USA, #101216), F4/80-FITC (Biolegend, San Diego, CA, USA, #123107), and the viability dye eFluorTM 506 (eBiosciences, San Diego, CA, USA, #65-0866).

After passage 3, harvested cells from the 10 samples of each group were washed with PBS and stained with monoclonal antibodies (McAb) for flow cytometry assay, which was performed according to MSC ISCT definition criteria [26 (link)]. The following McAb conjugated with either fluorescein isothiocyanate (FITC), phycoerythrin (PE), the tandem peridinin chlorophyll protein cyanine Cy5.5 (PerCP-Cy5.5), or allophycocyanin (APC) were used: CD34-FITC (Invitrogen); CD19-PerCP-Cy5.5, CD44-FITC, CD45-PerCP-Cy5.5, CD73-PE, CD90-FITC, CD166-PE, HLA-DR-PerCP-Cy5.5, (BD Biosystems); CD105-APC, CD106-FITC (R&D Systems); CD14-PE (Cytognos); and CD54-PE (Biolegend). 7-amino-actinomycin D (7AAD—Viability Stain solution, Biolegend) staining was employed to exclude dead cells (positive). Cells were acquired in a FACSCalibur flow cytometer (BD Biosystems) under a specific compensation and establishing an appropriate acquisition gate for forward- (FSC) and side scatters (SSC). Flow cytometry files were analyzed using Infinicyt^TM v.1.8 software (Cytognos). Both percentages of positive cells and Median Fluorescence Intensity (MFI) of each marker were measured and compared to unstained cells used as control.