Cd69 pe

In brief, healthy donors and patient’s total PBMC were stained for surface markers with the following fluorochrome conjugated antibodies: CD19-BUV737, CD107a-BUV395, CD3-BV786, CD33-BV711 & CD14-BV650 (for AML study), CD15-BV650 & CD30-BV605 (for HL study), PD1-FITC, CD62L-PerCP-Cy5.5, CD57-PE-CF594, CD16-AF700 from Beckton Dickinson, CD69-PE from Beckman Coulter, CD45RO-VioGreen, CD7-VioBlue, CD56-PE-Vio770, CD45RA-APC-Vio770, and NKG2C-APC from Miltenyi Biotec. Cell viability was determined using DAPI (BD Biosciences). Cells were stained with the corresponding antibodies in FACS buffer (PBS, 2% FBS) on ice for 25–30 min and washed twice with the same FACS buffer and acquired on BD LSR-Fortessa instrument (Blue-Yellow/Green-Red-Violet-Ultraviolet) (BD Bioscience). Flow Cytometry Standard (FCS) files were analyzed using FlowJo software v10.6.1 (Becton, Dickinson and Company, Ashland, OR). The gating strategy for conventional flow cytometry and the determination of the “percentage” of selected cells in the figures is described in the Supplemental Figure S1.

CIKs and MSCs were harvested, transferred into PBS, and incubated with Fc-solution (Beckman Coulter) for 5 min at 4°C to prevent unspecific antibody binding. During the incubation with Fc-blocking reagent, antibodies were placed in fluorescence-activated cell sorter (FACS) tubes (Greiner, BIO). The following antibodies, purchased from Beckman Coulter (final concentrations in brackets), were used: CD3-FITC (1∶100), CD4-PE (1∶250), CD8a-PE (1∶250), CD11b-PE (1∶160), CD19-PE (1∶250), CD25-PE (1∶250), CD45-PE (1∶250), CD49-PE (1∶250), NK1.1-PE (1∶60), CD69-PE (1∶250), HamIgG-FITC-isotype-control (1∶250), and RatIgG2a-PE-isotype-controll (1∶250). In addition, CD34-PE (Caltaq, 1∶100), CD44-AF488 (Biozol, 1∶250), CD73-PE (Biozol, 1∶100), CD90 (Thy1.2)-FITC (Miltenyi, 1∶100), and CD166 (Antikörper-online, 1∶100).
After incubation for 5 min, Fc-blocked cells were added to the antibody-containing FACS tubes, incubated for 20 min at 4°C, and washed with PBS. Then, 7-aminoactinomycin-D (7-AAD; 1∶100, Beckman Coulter) stain was added, and the cells were incubated for 5 min at 4°C. Either isotypes or autofluorescence served as controls. Cells were gated on vital cells, indicated by low 7-AAD signals.

Flow cytometry was performed using the following antibodies from BioLegend, unless otherwise stated. Antibodies to the following human molecules were used: CD69-AF647 (FN50), CD69-PE (FN50), CD3-BV421 (OKT3), γδTCR-PeCy7 (IMMU510; Beckman Coulter), CD45-PacificBlue (HI30), TCRVδ2-FITC (B6). Antibodies to the following murine molecules were used: TCRδ-PerCPe710 (GL3), TCRδ-APC (GL3), Vγ7-AF647 (F2.67, provided by P.Pereira). Other antibodies were as follows: DYKDDDDK-PE (FLAG, L5), HA-AF647 (16B12). 6xHis-APC (Biolegend, J095G46). Data were acquired on BD Canto II or Fortessa cytometers. sTCR staining was performed as in Melandri et al. (2018) (link).