Cd28 apc

Cells were stained for 30 min at 4 °C in PBS containing 0.01% NaN₃ and 0.5% BSA with directly labelled antibodies against: CXCR3 alexa fluor488, CCR5 PE, CCR4 PercP-Cy5.5, CCR7 PE-Cy7, CCR6 alexa647, CD4 APC-H7,CD8 V500, CD3 V500, CD69 PercP, CD45 V500, Foxp3 PercP-Cy5.5, Granzyme-A PE, CD8 V450 (all from BD Biosciences), CD3 FITC (Sanquin, Amsterdam, The Netherlands), CD4 PE-Cy7, CD28 APC, CD8 APC-efluor780, CD45RA efluor450, CD45RO PE (all from eBioscience Inc., San Diego, CA, USA), Granzyme-K Fitc (Immunotools, Friesoythe, Germany), Granzyme-B APC (Invitrogen, Thermo Fisher Scientific Inc.) and Perforin PercP-Cy5.5, IL-10 Pe-Cy7 (Biolegend, San Diego, CA, USA). For cytokine staining, we used the Th1/Th2/Th17 kit from BD Biosciences. Cells were analysed on an FACS Canto II (BD Biosciences) and data were analysed using the FlowJo software (FlowJo, Ashland, OR, USA).
Data were plotted as frequency of positive cells or as the gMFI to illustrate cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest was normalized to the gMFI of the negative population.

Mo/Ma were harvested and stained with anti-human CD14 AlexaFluor-647 or FITC, CD206 APC, Gal-9 PE, CD16 PE, CD40 PE-Cy5 (Biolegend), HLA-DR PE-Cy7, B7.H4 Biotin (eBioscience, San Diego, CA, USA) or isotype controls in PBS with 2% FSB on ice for 20 min. T cells were harvested and stained with mAb to human CD3 PerCP, CD27 APC-Alexa750, CD28 APC and Tim-3 PerCP-eFluor710 (eBioscience).
To detect CD40L expression, PE-labeled anti-human CD40L/CD154 antibodies were cultured with T cells in the presence of anti-CD3 mAb and Monensin solution (eBioscience) during 40 h. Intracellular staining for phosphotyrosine was performed an AlexaFluor-647-conjugated anti-human phosphotyrosine (PY20, Biolegend) antibody.
The production of ROS and NO was evaluated using the molecular probes: H₂DCF-DA (10 μM, Invitrogen Inc.) and DAF-FM DA (10 μM, Molecular Probes, Inc.), respectively.
The assessment of phagocytosis was performed using 1 μm-Fluoresbrite Yellow Green (YG) Carboxilate Microspheres (Polysciences, Inc., Warrington, PA, USA; 1 : 25 cell/microspheres ratio), which were added to CCs for 30 min. Afterwards, the uptake of YG-microspheres was evaluated in CD14+ cells.
All samples were acquired on a FACS Canto II (BD Biosciences, San Jose, CA, USA) and then analyzed with Flow Jo software (LLC, Ashland, OR, USA).