Hla dr alexafluor700

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).

For mCD40L expression in CFPAC-1 cells transduced with RAdnCD40L, 3 × 10⁵ cells were washed three times with ice-cold PBS buffer and incubated on ice for 20 min with 100 μl of diluted mouse anti-human CD40L-APC conjugate Ab or mouse isotype-APC Ab conjugate (ebioscience, San Diego, CA, USA) or left without treatment as negative control. Cells were then washed three times with ice-cold staining buffer (PBS with 1%BSA and 0.1% NaN3) analysed by flow cytometry. For monocyte-derived DC activation markers, mouse anti-human mAb CD1a- PE-Cy7, CD14-PE, CD40-APC, and HLA-DR-Alexa Fluor 700 were from ebioscience. For mouse anti-human mAb CD86-PE, CD83-APC and CD54-APC, were from BD bioscience, San Jose, CA, USA. Briefly, DC were stained with mouse anti-human mAbs or the isotype control for 20 minutes, cells were washed twice with staining buffer. Flow cytometry was performed by acquiring cells using BD LSR Fortessa cell analyser (BD Bioscience). A minimum of 50,000 HLA-DR + cells were acquired per sample and data were analysed by FlowJo software version X (Tree Star, Ashland, USA)