αcd56

Detection of Siglec-7 ligands by flow cytometry was performed as previously described (6 (link)). AML-derived samples were analyzed as previously described (17 (link)). In brief, non-specific antibody binding by Fc receptors was blocked using 100 μL of Fc Receptor Blocker (Innovex Biosciences, Richmond, CA, USA) and dead cells were excluded from analysis by staining with a Fixable Viability Dye (ThermoFisher). For the Siglec ligands staining, recombinant human Siglec-7-hFc (R&D Systems, Minneapolis, MN, USA) was pre-incubated with PE-conjugated goat anti-human Ig (Jackson ImmunoResearch Laboratories, West Grove, USA) for 1 h at 4°C and then applied to the samples for 1 h at RT together with lineage-associated antibodies. Lineage-positive cells were stained with biotinylated αCD2, αCD14, αCD16, αCD19, αCD56, and αCD235, together with fluorophore-conjugated αCD8, αCD4, αCD45, αCD34, αCD38 (all from BioLegend), followed by a second step with streptavidin-FITC conjugate (BD Biosciences). Cells were washed and analyzed on a BD FACSVerse or BD FACSlyric (BD Biosciences). Data were analyzed using the FlowJo 10.0.6 software (Tree Star Inc.).