Cd4 apc cy7 clone rpa t4

After culture, the cells were analyzed by flow cytometry for expression of CD23 or proliferation by dilution of CFSE. For detection of live cells, fixable viability dye e780 (Invitrogen) or live/dead Aqua stain (Molecular Probes) was used. Antibodies used were IgD-FITC and -BV421 (clone IA6-2), CD27-PE and -APC (clone M-T721), CD19-PE (clone HIB19), CD4-APC-Cy7 (clone RPA-T4), CD19-APC-H7 (clone SJ25C1), CD16-BV421 (clone 3G8), and CD56-BV421 (clone NCAM16.2) from BD Biosciences, and CD3-PerCP-Cy5.5 (clone HIT3a), CD23-APC (clone EBVCS-5), CD8-APC (clone HIT8a), and CD4-BV421 (clone RPA-T4) from BioLegend.
At least 10,000 live cells or 3,000 CD3-CD19⁺ events were collected on a FACSCanto II flow cytometer (BD Biosciences). Data were analyzed using FlowJo v 10 software (FlowJo Software for Windows).

After isolation by CD3 microbeads (see above), CD3⁺ T cells were stimulated with MSC or bmMSC preparations, in the respective culture medium (see above). Treg assay was performed in a 96-well round-bottom plate coated with antibodies against CD3 (10 µg/ml, clone OKT-3; ThermoFisher scientific) and CD28 (2 µg/ml, clone 28.2; Beckman Coulter) for T cell-activation. To assess the effect of MSC cells on CD3⁺ T cells, cells were co-cultured in a T-cell:MSC ratio of 2:1 (0.5*10⁵ CD3⁺: 0.25*10⁵ MSCs) at 37°C, 5% CO² To test effects of EVs isolated from stimulated MSC, 0,5*10⁵ CD3⁺ T cells were cultured in the present or absence of 30 µl EV preparations. After 3 days of culture CD3⁺ T cells were stained with CD4-APC-Cy7 (clone RPA-T4), CD25-APC (clone NM-A251, both BD-Bioscience), CD127-PE-Cy7 (eBioRDR5, ThermoFisher scientific), and intracellular with FoxP3-FITC (ECH101, both ThermoFisher scientific). Tregs induction were determined with marker expression of CD4⁺ CD127^dim CD25⁺ FOXP3⁺ of total CD4⁺. Cells were analysed with BD FACSCanto II using BD FACS DIVA 8.01 software (BD Biosciences).

For both the in vitro and ex vivo studies PBMC were activated for four hours with CD2/CD3/CD28 T cell activation beads (Miltenyi Biotec, Cologne, Germany) in the presence of GolgiStop and GolgiPlug protein transport inhibitors (BD Bioscience, Franklin Lakes, NJ), and anti-human CD107a APC conjugated antibody (clone H4A3, BD Bioscience). Cell surface marker staining was performed for the in vitro studies using LIVE/DEAD Aqua fixable dead cell stain (Thermo Fisher Scientific), CD4 APC Cy7 (clone RPA-T4, BD Bioscience), CD3 BV785 (clone OKT3, BioLegend, San Diego, CA) CD8α BV650 (clone RPA-T8, BioLegend), and IFNγ PE (clone B27, BD Biosciences). Cell surface marker staining was performed for the ex vivo study using LIVE/DEAD Aqua fixable dead cell stain, CD4 BUV395 (clone SK3, BD Biosciences), CD3 BUV496 (clone UCHT1, BD Biosciences) and CD8α BUV805 (clone SK1, BD Biosciences). Surface and intracellular cytokine staining was performed using the Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer’s instructions.