Cytkick max autosampler

Freshly thawed PBMCs (3.0 × 10⁵ cells per well) were dispensed into a U-bottomed 96-well plate in 100 µl of RPMI with 10% non-heat-inactivated human serum (Cat: H4522-20Ml; Sigma-Aldrich) and left unstimulated or they were stimulated by incubation with the following reagents for 18 h: Dynabeads Human T-Activator CD3/CD28 beads (Cat: 11131D; Gibco, bead:cell = 1:1), ImmunoCult Human CD3/CD28/CD2 T-Cell Activator (Cat: 10970; StemCell Technologies, final dilution 1:100), PHA-M (Cat: 10576015; Gibco, final dilution 1:100), and Cell Stimulation Cocktail (Cat: 00-4970-93; eBioscience, final 1:1,000). For blinatumomab-mediated T cell activation, freshly thawed PBMCs (1.0 × 10⁵ cells per well) were dispensed into a U-bottomed 96-well plate in 100 µl of RPMI with 10% of non-heat-inactivated human serum and rhIL-2 (1:500). They were left unstimulated or were stimulated with blinatumomab (BPS Biosciences, Cat: 100441-2, final concentration 10 ng/ml) for 3 or 5 d. Supernatants were harvested and analyzed with the LEGENDplex HU Th Cytokine Panel V02 kit (Cat: 741027; BioLegend). Beads were acquired with an Attune NxT Flow Cytometer with the CytKick MAX Autosampler (Invitrogen).

HuT78 cells (1 × 10⁵ cells per well) and Raji cells (1 × 10⁵ cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen). Monensin and brefeldin A (1:1,000 each; Cytek) were added for the last 6 h. Cells were stained with Zombie NIR Fixable Viability dye (1:1,000 in PBS; BioLegend) for 15 min at 4°C in the dark, washed with FACS buffer, and fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (Cytek). Cells were then stained by incubation overnight at 4°C in the dark with the following reagents in permeabilization buffer: FcR blocking reagent (1:50; Miltenyi Biotec), anti-CD3-APC (Clone: UCHT1, 1:100; Cytek), anti-IFN-γ-PE-Dazzle 594 (Clone: 4S.B3, 1:500; BioLegend), and anti-TNF-BV711 (Clone: MAb11, 1:500; BioLegend) mAbs. The cells were washed with FACS buffer and acquired with an Attune NxT Flow Cytometer with the CytKick MAX Autosampler (Invitrogen). Data were analyzed with FlowJo and R software. The percentage of IFN-γ⁺ cells was used as a readout. The data were normalized against the mean for the blinatumomab plus atezolizumab group for each combination of HuT78 and Raji cells.