Ebioscience fixation permeabilization kit

Blood from buffy coats was added to red blood cell (RBC) lysis buffer (BioLegend), and after washing, 1 × 10⁶ leukocytes were incubated with FcR blocking (Miltenyi Biotec) for 10 min at 4°C. Cells were stained with mAbs CD14 Pacific Blue (63D3), CD177 APC/Cy7 (MEM-166), CD19 PE/Cy7 (HIB19), CD4 Alexa Fluor 488 (OKT4), and CD8 APC (HIT8a) (all from BioLegend), fixed, and permeabilized with the eBioscience fixation/permeabilization kit (Thermo Fisher Scientific).
Intracellular staining was performed with rabbit anti-SSC4D polyclonal antibody, followed by anti-rabbit PE labeling (Life Technologies). Data were acquired in the FACSCanto II and post-acquisition analysis performed using FlowJo v10.
For cell sorting, blood from buffy coats was added to RBC lysis buffer, and 1 × 10⁷ leukocytes were stained with mAbs CD14 APC (63D3), CD177 APC/Cy7 (MEM-166), CD19 PE/Cy7 (HIB19), and CD3 PerCP/Cy5 (OKT3). The labeled cells were sorted with FACSAria (BD Biosciences).

If not specified otherwise, cells were stained for functional and phenotypical markers and analyzed via flow cytometry using an LSR Fortessa II (BD). When specified, cells were analyzed using mass cytometry (CyTOF) with a Helios mass cytometer (Fluidigm, South San Francisco, CA, USA). When the antibodies required conjugation, MaxPar X8 labeling kits (Fluidigm) were used according to the manufacturer’s protocol. Cells were stained with viability marker, LIVE/DEAD Fixable Near-IR (Invitrogen, Waltham, MA, USA) or Fixable Viability Dye eFluor 780 (Thermofisher, Waltham, MA, USA), which was followed by Fc-receptor blocking with TruStain fcX (Biolegend) for mouse experiments and extracellular staining. For intracellular staining, cells were treated with the eBioscience Fixation/Permeabilization kit (Thermofisher). To obtain the HLA-E expression of target cell lines, target cells from culture were stained with viability marker LIVE/DEAD Fixable Near-IR (Invitrogen), followed by staining for HLA-E expression. Detailed information about the antibodies used can be found in Table A2 and Table A3 for mass and flow cytometry, respectively. Data were analyzed using FlowJo (BD Biosciences; v.10 or later).