Anti cd69 fitc

To analyze the expression of the activation markers CD69 and CD25 on the surfaces of T cells, 1 × 10⁴ transgenic CD33-expressing HEK293T cells were co-cultured with 5 × 10⁴ untouched pan T cells in the presence or absence of 48 h cultured 1 × 10⁴ SCP-1 cells. At the indicated time points, triplicate cells were pooled and stained with a mixture of anti-CD3/VioBlue, anti-CD69/FITC, anti-CD4/PerCP (all purchased from Miltenyi Biotec) and anti-CD25/PE (BD Biosciences) mAbs. T-cell proliferation assays were performed as described.^{25 (link), 28 (link)}

For immunophenotyping, cells were stained in PBS supplemented with 0.5% BSA and 2mM EDTA for 15min at 4°C with following fluorochrome-conjugated antibodies: anti-CD69-FITC or anti-CD3-FITC, anti-TCR Vα7.2-PE, anti-CD161-APC (all from Miltenyi Biotec, Germany), anti-CD223(LAG3)-FITC, anti-CD154(CD40L)-FITC (Biolegend, USA). Dead cells were excluded using propidium iodide (Miltenyi Biotec, Germany). MAIT cells were identified based on their TCR Vα7.2 and CD161 expression. Cell surface expression was analyzed on a FACS Calibur (BD Bioscience). Cell sorting was carried out using FACS ARIA II (BD Bioscience). Data were analyzed using FlowJo™ Software Version 10.5.3 (Becton, Dickinson and Company).

Freshly isolated human pan T-cells co-incubated with 1 × 10⁴ CD33⁺ MOLM-13 cells at an effector-to-target (E:T) cell ratio of 1:1 in the presence or absence of 1 × 10⁴ bsAb-releasing MSCs seeded either in 2- or 3D were analyzed for the surface expression of specific T cell activation markers CD25 and CD6922 . After 48 h of co-culture, the cells of one triplicate were pooled and stained with a mixture of anti-CD3/VioBlue, anti-CD69/FITC (all purchased from Miltenyi Biotec) and anti-CD25/PE (BD Biosciences, Heidelberg, Germany) mAbs.