Macsquant analyzer vyb

Cytotoxicty assays were performed as described previously (60 ). Target cells were left unpulsed or pulsed with 10 μM antigenic peptide for 2 hours at 37°C/5% CO₂, washed three times, and subsequently transferred to a 96-well U-bottom plate at 20 × 10³ cells per 100 μl of RPMI 1640 5% FCS/Hepes. CTLs were previously stained with 0.1 μM CMFDA for 30 min at 37°C/5% CO₂, washed, and added to the target cells at a four CTL/one target cell ratio in 100 μl of RPMI 1640 5% FCS/Hepes. Cells were pelleted for 1 min, 455g and incubated at 37°C/5% CO₂ for 4 hours. In some experiments, CTLs were pretreated with 40 μM monensin (Sigma-Aldrich) and thoroughly washed before conjugation with target cells or pretransfected with perforin siRNA. In additional experiments, melanoma cells were pretreated with 50 μM BAPTA-AM (Invitrogen) and thoroughly washed before conjugation with CTL. In a supplementary set of experiments, D10 cells were previously transfected with a plasmid coding for SytVII shRNA. Before flow cytometry analysis, 0.25 μg of 7-aminoactinomycin D (BD Biosciences) was added to each sample to measure the percentage of dead target cells. Samples were acquired using MACS Quant Analyzer VYB (Miltenyi Biotec). Results were analyzed using the FlowJo 10 software.

MACSQuant Analyzer, MACSQuant Analyzer VYB (Miltenyi Biotec), or FACSCanto, LSRII (Becton Dickinson, NJ) was used to analyze cell populations by flow cytometry. The following antibody and protein conjugates were used: CD3-PE, CD3-APC-Vio770, CD4-APC, CD4-VioGreen, CD8-APC-Vio770, CD8-VioGreen, CD14-APC, CD16-PE, CD19-APC, CD20-PerCP-Vio770, CD20-PE-Vio770, CD34-APC, CD34-PE, CD45-VioBlue, CD45-VioGreen, CD45RO-PE-Vio770, CD56-PE, CD62L-VioBlue, CD95-APC, CD45-VioBlue (mouse) (all from Miltenyi Biotec); ErbB2-Fc fusion protein (R&D Systems), anti-human-IgG (Fc gamma-specific) PE (eBioscience). 7AAD staining was used for dead cell exclusion and Violet Cell Trace (Thermo Fisher) for SupT1 cell tracking.

Peripheral blood from healthy donors was obtained from the Red Cross Blood Center (Daejeon, Republic of Korea) according to established guidelines. The methods and protocols used in this study were approved by the Institutional Review Board of the Red Cross, and written informed consent for study participation was obtained from donors. Donor information was not disclosed. For the T cell proliferation assay, human T cells were isolated at >95% purity from peripheral blood based on negative selection using the RosetteSep Human T Cell Enrichment Cocktail (STEMCELL Technologies, Vancouver, Canada). Isolated human T cells were labeled with various concentrations of Carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, USA) at 37°C for 15 min. The labeled cells were collected, washed with fresh medium, counted, seeded in 96-well plates (1×10⁵ cells/well), and stimulated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies (Invitrogen) at a 1:1 cell:bead ratio. After 96 hours, the cells were collected and stained with 7-AAD (20 min at room temperature). Samples of unlabelled and stimulated cells were stained with anti-CD45-APC mAb (BD Biosciences) and anti-IFNγ-PE mAb (BD Biosciences). Immediately after this step, the cells were analyzed using a flow cytometer (MACSQuant Analyzer VYB, Miltenyi Biotec).

Cells are washed by PBS, lifted by 0.25% EDTA-Trypsin (Gibco), and diluted in HBSS (Gibco) with 0.25% of BSA before flow cytometry. Flow cytometry experiments are performed either on MACSQuant VYB Analyzer (Miltenyi Biotec) or CytoFLEX (Beckman Coulter). Analysis of data is done with open source, in-house developed software, EasyFlow (https://github.com/AntebiLab/easyflow) or EasyFlowQ (https://github.com/ym3141/EasyFlowQ).
FACS is performed with SY3200 Cell Sorter (Sony) at Caltech FLow Cytometry Facility.