Anti cd3 ab okt3 clone

GFP-expressing P815 target cells were coated with the indicated concentrations of anti-CD3 Ab (OKT3 clone, eBioscience) during 1 h at 37 °C/5% CO₂. The target cells were washed twice in PBS after coating. CD8⁺ T cells were stained with 2.5 µM CellTraceViolet (Thermo Fisher) during 10 min. P815 target cells and CD8⁺ T cells were transferred (2500 P815 cells and 5000 CD8⁺ T cells) in the wells of a 384-well plate (Perkin Elmer) precoated with 1 µg/ml fibronectin. Aphidicolin (Sigma) was added at 0.1 µg/ml to prevent target cell proliferation and allow net measurement of CD8⁺ T cell-mediated cytotoxicity. Well content was recoded every hour for 24 h with an automated high-content confocal microscope (Opera Phenix, Perkin Elmer) set to 37 °C and 5% CO₂. Cytotoxicity was analyzed by counting the number of alive target cells as detailed previously⁶⁰ .

P815 target cells were coated with the indicated concentrations of anti-CD3 Ab (OKT3 clone, eBioscience) during 1 h at 37 °C/5% CO₂. The target cells were washed twice in PBS after coating. CD8⁺ T cells were stained with CMFDA during 30 min. P815 target cells and CD8⁺ T cells were transferred at a 2:1 ratio in the wells of a 8-wells plate IBIDI precoated with 10 µg/ml fibronectin. Propidium iodide was added at 100 nM in order to visualize the apoptosis. Well content was recoded every minute by a spinning disk microscope equipped with temperature and CO₂ control (37 °C/5% CO₂). The formation of conjugates was studied with the 488 and brightfield channels in superposition, to detect CD8⁺ T cells and P815 target cells, respectively. Each time a CD8⁺ T cell contacted a P815 cell, an image crop was done to isolate the event and the contact time was determined manually with the time-points identified on the stacks.

GFP-expressing P815 target cells were coated with the indicated concentrations of anti-CD3 Ab (OKT3 clone, eBioscience) during 1 h at 37 °C/5% CO₂. The target cells were washed twice in PBS after coating. They were co-cultured with CD8⁺ T cells in 96-wells flat plate for 2 h at 37 °C/5% CO₂ (30,000 P815 cells and 60,000 CD8⁺ T cells). Anti-LAMP1 Ab (PE, BD Biosciences) has been added from the outset of the co-culture, and m24 Ab (Af647, Biolegend) and viability dye (APC-H7, eBioscience) has been added to the wells 20 min before the end of the experiment. The CD8⁺ T cells were stained with an anti-CD8 Ab (PerCP Cy5.5, Sysmex) for 20 min. Samples were acquired on Fortessa (BD Biosciences) and analyzed with FlowJo software (FlowJo, Ashland, Ore). The cells were gated in order to study alive (viability dye negative cells) and CD8⁺ T cells only.