Live deadtm fixable blue dead cell stain kit

Single cell suspensions of passage three or four were prepared using 0.05% Trypsin-EDTA (Gibco). To avoid unspecific antibody binding to Fc receptors (FcR), cells were blocked with FcR Blocking Reagent (Miltenyi Biotech) according to the manufacturer’s instruction. Cell surface marker were stained for 10 min using the antibodies and dilutions given in Table 3. LIVE/DEAD^TM Fixable Blue Dead Cell Stain Kit (Invitrogen) was used to exclude dead cells. Cells were acquired using a CytoFLEX LX flow cytometer (Beckman Coulter). Flow cytometric data were analysed with FlowJo V10 (FlowJo, LLC).

Stimulated PBMCs were washed with PBS and stained with Fc receptor blocking reagent (Miltenyi Biotec) and LIVE/DEAD^TM Fixable Blue Dead Cell Stain Kit (Invitrogen). Cells were washed with FACS buffer and stained for extracellular surface marker. Cell fixation and permeabilization were performed using BD Cytofix/Cytoperm^TM (BD Biosciences) following intracellular staining for cytokines and cytolytic molecules. Cells were washed twice with permeabilization buffer and cell pellet was resuspended in FACS buffer and subsequently analyzed on BD LSR-II SORP and BD LSR-Fortessa flow cytometer. Data analysis was then carried out by FlowJo (TreeStar, v10.4.2) and Prism (GraphPad Software, v7.0c). To determine significant differences, Wilcoxon matched-pairs signed rank test was used.

For cytometric assessment of MAIT cell phenotype, bulk PBMCs were stained with LIVE/DEAD^TM Fixable Blue Dead Cell Stain Kit (Invitrogen) and Fc receptor blocking reagent (Miltenyi Biotec) and a combination of the following antibodies (from BioLegend except as noted): CD3 BV655 (clone OKT3), CD8 BV711 (clone RPA-T8), CD161 APC (clone DX12, BD Biosciences), Vα7.2 (clone 3C10), CD69 PE (clone FN50), CD107a PerCP-Cy5.5 (clone H4A3), granzyme B Pacific Blue (clone GB11), perforin FITC (clone), and interferon γ APC-Cy7 (clone 4S.B3).