Nkp46 pe

HSPC-NK cell phenotype was determined by staining for CD56-PE-Cy7 (Biolegend, #318318), NKG2A-APC (Beckman Coulter, #A60797), CD16-FITC (Biolegend, #302006), pan-KIRs-PE (Biolegend, #339506, 312606 and 312708), DNAM-1-FITC (BD, #559788), NKp46-PE (Biolegend, #331908) and NKG2D-APC (Biolegend, #320808) (CellGro vs NK MACS) or CD56-BV510 (Biolegend, #318340), CD45-BV421 (Biolegend, #368522), NKG2A-PE-Cy7 (Beckman Coulter, #B10246), DNAM-1-FITC (Biolegend, #337104), NKp46-PE (Biolegend, #311908), NKp44-PE (Biolegend, #325108), NKp30-APC (Biolegend, #325210) and NKG2D-APC (Biolegend, #320808) (GMP validations runs). Briefly, 200.000 cells were washed using PBS/0.5% BSA and incubated with antibodies in PBS/0.5% BSA at 4 °C for 30 min. Cells were then washed twice with PBS/0.5% BSA and resuspended in PBS/0.5% BSA containing Sytox Blue (1:5000 diluted, invitrogen, #S34857) for CellGro vs NK MACS experiments or 7-AAD (1:1000 diluted, Sigma, #A9400) for GMP validation runs. Cells were acquired on the Gallios (CellGro vs NK MACS) or Navios (GMP validation runs) flowcytometers and analyzed using Kaluza V2.1.3.

MNCs were stained with labeled antibodies, CD3 ECD (Biolegend), CD45 Krome Orange (R&D systems), CD56 PE-Cy5 (Biolegend), CD16 APC-Cy7 (Biologend), CD326 PerCPCy5.5 (Biolegend). Phenotypic analysis was performed using DNAM-1 FITC (Becton Dickinson), 2B4 FITC (Biolegend), NKG2A APC (Beckman Coulter), NKG2D APC (Biolegend), NKp30 PE (Biolegend) and NKp46 PE(Biolegend), isotype controls for IgG1 and IgG2a, (all Biolegend). Dead cells were stained with 1:1000 diluted sytox blue (Life Technologies; Invitrogen). Flow cytometry analysis was performed on a Gallios flow cytometer from Beckman Coulter. Analysis was done in Kaluza 1.5 (Beckman Coulter). Gating strategy is shown in Supplementary Figure 1.

After co-culture, we evaluated the expression of NKp30, NKp44, NKp46, and NKG2D in NK cells by FC. First, NK cells were stain for viability using LIVE/DEAD Fixable Near-IR, after that NK cells were harvested, resuspended in PBS, and stained with CD56-APC, NKp30-PE, NKp44-PE, NKp46-PE, or NKG2D-PE (BioLegend); subsequently, the cells were incubated in the dark for 30 min at room temperature. Cells were washed and fixed with paraformaldehyde 1%. An appropriate isotype and FMO controls were utilized to adjust for background fluorescence, and results are reported as the % of expression or geometric Mean fluorescence intensity (MFI). For each sample, at least 10,000 events were acquired in a FACSAria I cell sorter (BD Bioscience). Data were processed with FlowJo ver. X.0.7 software (Tree Star, Inc.).