Cd33 fitc

CD157 surface expression on AML samples and cell lines was assessed by flow cytometry (BD FACS Canto, BD Biosciences) by staining cells (3 × 10⁵/sample) with a PE-labeled mAb against CD157 (clone SY11B5, Invitrogen, Milan, Italy) for 20 min at 4 °C. Leukemic blasts were analyzed by multiparametric flow cytometry and gated based on CD45 expression (anti-CD45-APC/H7, BD Biosciences) and side scatter (CD45^dim and SSC^low). To further characterize blast subpopulations the following antibodies were used: CD33-FITC, CD34-PE/Cy7, CD64-PE, CD117-PE, CD123-PE/Cy7, HLA-DR-FITC (Biolegend). Specifically, this back-gating allowed us to identify leukemic myeloid blasts (CD33^dim, CD64−, CD117+, CD123+, HLA-DR+) and leukemic monocytic populations (CD33+, CD64++, CD117^low/neg, CD123+, HLA-DR++). Data were analyzed using FlowJo software Version 10.6 (BD Biosciences). CD157 relative Mean Fluorescence Intensity (MFI) was normalized to the MFI of lymphocytes, which are CD157 negative. CD157 MFI ratio was calculated according to the formula: (MFI of CD157 in blasts − MFI of CD157 in lymphocytes)/MFI of CD157 in lymphocytes.

The following anti-human antibodies were used for the lineage the cocktail: CD3-FITC, CD4-FITC, CD8a-FITC, CD14-FITC, CD15-FITC, CD16-FITC, CD19-FITC, CD20-FITC, CD33-FITC, CD34-FITC, FcεRI-FITC, and CD203c-FITC (all from Biolegend). In addition, these anti-human antibodies were used: CD335 (NKp46)-PE/Cy7, CD294 (CRTH2)-PE, CD127-BV421 from Biolegend, CD56-APCeF780 from eBioscience, and CD117 (ckit)-APC from BD Bioscience. For intracellular transcription factor staining the following anti-human antibodies were used: Tbet-PE/CF594, GATA3-PE/Cy7, and RORγt-PE from BD Bioscience. Corresponding isotype control antibodies were used as controls.