Apc conjugated anti human cd3

Whole blood was taken from healthy donors. Using Ficoll–Paque density gradient centrifugation, human peripheral blood mononuclear cells (PBMCs) were isolated. Isolated PBMCs were seeded into 24-well plates (1.5 × 10⁶ cells/well) and cultured in RPMI1640 containing 10%FBS and 100 IU hIL-2 (Miltenyi Biotec). To activate and enrich T cells, PBMCs were cultured with 3 ug/ml anti-CD3 antibody (Miltenyi Biotec) and 10 ug/ml anti-CD28 antibody (Miltenyi Biotec). After 4 days of incubation at 37 °C, the purity of T cells was assayed using APC conjugated anti-human CD3 (BioLegend, USA) by flow cytometry. The clone of APC anti-human CD3 Antibody was UCHT1.The purity of T cells is represented in the figure S 2.

Brilliant Violet 421-conjugated anti-human CD45 (HI30), APC-conjugated anti-human CD3 (OKT3), APC-conjugated anti-human CD19 (HIB19), FITC-conjugated anti-human CD14 (M5E2), CD11c-conjugated anti-human CD11c (3.9), PE-Cy7-conjugated anti-human HLA-DR (LN3), PerCP-Cy5.5-conjugated anti-human CD56 (5.1H11), PE-conjugated anti-human LILRB4 (ZM4.1), Pacific blue-conjugated anti-mouse CD45 (30-F11), Brilliant violet 510-conjugated anti-mouse CD11b (M1/70), PerCP-Cy5.5-conjugated anti-mouse CD11c (N418), FITC-conjugated anti-mouse Ly6G (1A8), PE-Cy7-conjugated anti-mouse CD64 (X54-5/7.1), APC/Fire 750-conjugated anti-mouse CD24 (M1/69), APC-conjugated anti-mouse IA/IE (M5/114.15.2), PE-conjugated anti-mouse LILRB4 (H1.1), PE-conjugated American Hamster IgG control antibody (HKT888) were purchased from Biolegend. APC conjugated anti-human CD206 (19.2), APC-conjugated mouse IgG₁κ control antibody (P3.6.2.8.1), PE-conjugated mouse IgG₁κ control antibody (P3.6.2.8.1) were purchased from eBiosciences.

PBMCs were plated in 35 mm non-treated dishes (2 × 10⁶ cells per dish) in 2 mL of CM harvested from hADSCs-IL2, native hADSCs or fresh DMEM/F12. Activation of PBMCs populations was determined at 72 h by flow cytometry using FACS Aria III (BD Biosciences, San Jose, CA, USA), a minimum of 20,000 events were acquired for each sample. The following populations of the immune cells were investigated: late-activated T-cells (CD3⁺, HLA-DR⁺), activated CD8⁺ T-cells (CD8⁺, CD38⁺), early-activated T-cells (CD3⁺, CD25⁺), NK cells (CD3⁻, CD56⁺), NKT-cells (CD3⁺, CD56⁺). PBMCs were washed twice with PBS and resuspended at 1 × 10⁵ cells/mL in PBS before being incubated with antibodies for 30 min in the dark at RT. All samples were stained with allophycocyanin (APC)-conjugated anti-human CD3 (#300312, BioLegend, San Diego, CA, USA) or Alexa Fluor 488-conjugated anti-human CD3 (#2324020, Sony Biotechnology, San Jose, CA, USA), Alexa Fluor 488-conjugated HLA-DR (#IM0463U, Beckman Coulter, USA), Alexa Fluor 647-conjugated anti-human CD56 (NCAM) (#2191564, BioLegend, San Diego, CA, USA), R-phycoerythrin (PE)-Cy7-conjugated anti-human CD25 (#2113060, Sony Biotechnology, San Jose, CA, USA), PE-conjugated anti-human CD8a (#300908, BioLegend, San Diego, CA, USA) and Alexa Fluor 488-conjugated anti-human CD38 (#2117515, Sony Biotechnology, San Jose, CA, USA).