Cd123 pe cy7

Neutrophils or PBMC at concentration 10⁶/mL were stimulated with LPS, ssRNA, R848, or polyI:C overnight and the cytokine levels in the culture supernatants were determined by multiplex Luminex cytokine bead-based immunoassay (R&D Systems).
For intracellular cytokine detection, 100 uL of peripheral blood from EDTA-coated tubes was stained against lineage-specific markers (CD3, CD19, CD20, CD56) conjugated with FITC, CD16-A700, CD11c-APC, CD14-PE-DyLight594 (Exbio), HLA-DR-PerCP (BD Biosciences), and CD123-PE-Cy7 (BioLegend). After RBC were lysed with BD Lysing solution (BD Biosciences), cells were fixed and permeabilized using a FixPerm kit (Thermo Fisher Scientific). Cytokines were stained using anti IL-6-PE (clone MQ2-13A5) (Biolegend), IL-1β-PE (clone CRM56) (Thermo Fisher Scientific), and TNFα-PE (clone MAb11) (Exbio), respectively. FMO controls were used to determine the cells producing the analyzed cytokines.

Samples were resuspended in FACS buffer (2% FBS, 0.1% sodium azide) and stained with the following mixtures of Biolegend antibodies: BDCA1-Percp/Cy5.5, CD14-APC, HLADR-Pacific Blue, CD3/19/56-FITC, CD123-PE/Cy7, and either FcεRIα-PE or mouse IgG_2b-PE, all at manufacturer's recommended concentrations. Some samples were stained with CD45-A700 to identify hematopoietic cells and CD203c-biotin (Biolegend, at manufacturer's recommended concentrations) followed by streptavidin-A647 (Invitrogen, at manufacturer's recommended concentration) to identify mast cells. Cells were stained with antibodies and propidium iodide (PI) (Biolegend) at 1∶400 for 15 minutes at 4 degrees, washed and spun at 1300 rpm, and resuspended in FACS buffer. At least 1×10⁶ cells were run on slow or medium speed on a BD LSRII machine and analyzed with Flowjo software.

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.