Cd34 pe cy7

Electroporated CD34⁺ HSPCs were stained with CD45-AlexaFluor700 (BioLegend, clone H130), CD34-PE/Cy7 (BioLegend, clone 581), CD44-BV421 (BioLegend, clone IM7), and CD29-APC (BD Pharmingen, clone MAR4 (RUO)). Mouse BM cells were stained with CD45-AlexaFluor700 (BioLegend, clone H130), HLA-A,B,C-PE/Cy7 (BioLegend, clone W6/32), CD33-PE (BioLegend, clone WM53), and CD19-BV605 (BD Biosciences, clone SJ25C1). All antibodies were used in the concentration recommended by the manufacturer. Dead cells and debris were excluded by forward scatter (FSC), side scatter (SSC), DAPI or 7-AAD staining (1:100 dilution prior to analysis). The flow cytometry analyses were performed with BD LSR II instrument (BD Biosciences) or BD LSRFortessa instrument (BD Biosciences). To analyse the data, the software FlowJo (FlowJo, LLC) was used.

To assess the pluripotency of iPS cells, the antibodies TRA1-60-PE (Cat Nr. MA1-023-PE, eBioscience) and SSEA4-FITC (Cat Nr. 560126, BD biosciences, BD) were used. Dead cells were excluded from the analysis by 4′,6-diamidino-2-phenylindole (DAPI; 1μg/ml) (Cat Nr. D3571, Thermo Fisher Scientific) staining. For detection of hematopoietic progenitor cells, the antibodies CD33-BV421 (Cat Nr. 366622, BioLegend, BL), CD34-PE-Cy7 (Cat Nr. 343615, BL), KDR-AF647 (Cat Nr. 359909, BL), CD43-PE (Cat Nr. 343204, BL), CD41a-FITC (Cat Nr. 303703, BL), CD235a-FITC (Cat Nr. 349103, BL), CD45-BV510 (Cat Nr. 103138, BL) and 7-AAD (Cat Nr. 420404, BL) were used as an “early-stage” multicolor hematopoietic cell panel. For the detection of mature myeloid cells, the antibodies CD15-PE (Cat Nr. 301905, BL), CD16-FITC (Cat Nr. 302005, BL), CD14-APC-H7 (Cat Nr. 367117, BL), CD45-BV510 (Cat Nr. 103138, BL), CD33 BV-421 (Cat Nr. 366622, BL) and 7-AAD (Cat Nr. 420404, BL) were used as a “late-stage” multicolor myeloid differentiation panel. Anti-mouse IgGk beads were used for compensation. Antibodies and beads for flow cytometry were purchased from BD Biosciences unless otherwise indicated. Samples were analyzed using a FACS Canto II flow cytometer (Becton-Dickinson) and FlowJo software (FLOWJO, LLC, Ashland, OR).

In brief, lungs were isolated from mice and cut into small pieces. The fragments were digested in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1% collagenase A (Roche)/0.1% dispase (Roche), and 10 U/ml DNase I (Roche) at 37°C for 60 min. The lung fragments were filtrated through a 70‐µm Cell Strainer (Greiner Bio‐One) into phosphate‐buffered saline, containing 5 mM ethylenediaminetetraacetic acid and 0.5% FBS. The single cell suspensions were divided into 1 × 10⁶ cells each per analysis and underwent a flow cytometric analysis with a FACS Versa (BD Biosciences‐Pharmingen). For the staining, fluorescence‐conjugated monoclonal antibodies against the following targets were used at a concentration of 0.25 µg/10⁶ cells in 100 µl volume: CD45 APC/Cy7 or PerCP/Cy5.5, CD11b PECy7 or PerCP/Cy5.5, CD11c PECy7, Gr‐1 PECy7 or APC, CD34 PECy7, CD40 PECy7, CD64 PECy7, CD80 PECy7, CD86 PECy7, IA/IE PECy7, F4/80 PECy7 or APC, CXCR4 biotin, Sca‐1 PECy7, Ly6C PECy7, Ly6G PECy7, MerTK PECy7, all from BioLegend, as were 7‐AAD Viability Staining Solution and PECy7‐conjugated Streptavidin. Each analysis of flow cytometry was performed through three experiments with a total of six mice per group.

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.