THP-1/TIM-3-Flag cells (TIM-3 over-expressers) were labeled with 2 μM CFSE (Carboxyfluorescein succinimidyl ester, Thermo Fisher Scientific), and THP-1 parental cells were labeled with 2 μM CTV (Thermo Fisher Scientific), according to the manufacturer’s instructions. THP-1 cells were mixed at a 1:1 ratio for a total of 100,000 THP-1 cells per well (50,000 THP-1 TIM-3-Flag and 50,000 THP-1 parental cells) and co-cultured for 3 days with 100,000 T cells purified using a human pan T-cell isolation kit (Miltenyi Biotec) from healthy human donor peripheral blood mononuclear cells (PBMCs) (Bioreclamation), in the presence of varying amounts of anti-CD3/anti-CD28 T-cell activation beads (Thermo Fisher Scientific) and 25 μg/ml sabatolimab whole antibody, sabatolimab Fab, sabatolimab Fab’2 or hIgG4 isotype control. On day 3 (~72 hours) of co-culture, cells were detected and counted by flow cytometric analysis. Differences in the killing of THP-1/TIM-3-Flag cells and THP-1 parental cells were determined by calculating the ratio of remaining cells at the end of the experiment.
Anti cd3 anti cd28 t cell activation beads
Manufactured by Thermo Fisher Scientific
Anti-CD3/anti-CD28 T-cell activation beads are a type of laboratory equipment used for stimulating and expanding T cells in cell culture. These beads are coated with antibodies that bind to the CD3 and CD28 receptors on the surface of T cells, triggering their activation and proliferation.
Automatically generated - may contain errors
2 protocols using anti cd3 anti cd28 t cell activation beads
1
TIM-3 Dependent T Cell Killing Assay
2
Mesenchymal Cell Activation by Treg Cells
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Mesenchymal cells were isolated and plated as for RNA-Seq. Briefly, Foxp3gfp mice were infected with 400 TCID50 PR8/H1N1 7 d prior to mesenchymal cell isolation. After plating mesenchymal cells overnight at 50,000 cells/well, Treg cells were FACS-sorted from Foxp3gfp mice at dpi 8 and co-cultured with mesenchymal cells for 12 h at a 1:2 ratio (Treg:Mes) in the presence of anti-CD3/anti-CD28 T cell activation beads (Thermo Fisher Scientific, Dynabeads Mouse T-cell Activator). For stimulation experiments and controls, rmEGF was used at 100 ng/ml and rmAREG was used at 200 ng/ml. At the end of each incubation, wells were washed once with EDTA to remove Treg cells, then twice with PBS, followed by lysis of mesenchymal cells in Trizol for RNA isolation and qPCR.
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