Cd13 pe

Cells (seeding density 2 × 10⁵ in each well/6-well plate) were pelleted by centrifugation for 5 min at 500× g. The supernatant was removed, and cells were resuspended in 10 μL of CD133 (APC Mouse Anti-Human CD133), CD13 (CD13 PE), and CD24 (Alexa Fluor^® 700 Rat Anti-Mouse CD24) antibodies (BD, Berkshire, UK) and incubated for 30 min on ice in the dark. Then cells were washed twice with 1× PBS for 5 min at 500× g and resuspended in 200 μL of 1X PBS. Results were analyzed by a flow cytometer using C6 software (BD LSRFortessa X-20; BD, Berkshire, UK).

To investigate cellular proliferation, DPSCs were labelled with 5 μM of 5-chloromethylfluorescein diacetate (CMFDA) in α-MEM for 45 min at 37°C CO₂. Cells were washed in medium and seeded at 10⁵/well in a 12 well-plate, in duplicate, for different time points (2, 3, and 4 days). Cytoplasmic amount reduction of the dye was measured using NAVIOS flow cytometer (Beckman Coulter, Brea). The data were analysed with FlowJo software. For immunophenotype analysis, cells cultured for 1 week with 10% FBS or 1% PL were trypsinized and aliquoted in FACS tube. Cells were washed twice with PBS 0,1% BSA. To limit unspecific binding, a blocking step is performed by resuspension of the pellets with PBS 1% BSA for 15 min. Cells were stained on ice for 1 h with saturating concentrations of primary conjugated antibodies diluted 1 : 50 in PBS 0,1% BSA. CD13-PE (mouse IgG1), CD29-APC (mouse BALB/c IgG1), CD44-FITC (mouse IgG2b), CD45-APC-H7 (mouse IgG1), CD73-FITC (mouse IgG1), CD90-PE (mouse BALB/c IgG1), and CD105-APC (Mouse BALB/c IgG1) monoclonal antibodies purchased from BD (Franklin Lakes) and CD146-PE (mouse IgG1), CD34-FITC (mouse IgG2a), and HLA-DR-PE (recombinant human IgG1) monoclonal antibodies purchased from Miltenyi Biotec (Bergisch Gladbach) were used to define the MSC panel as previously described [15 (link)]. At least 10000 events were counted for each sample.

For analysis of surface markers, cells were stained in PBS containing 2% BSA. The following fluorescent‐labeled antibodies (purchased from BD Biosciences) were used: CD13 PE (652820), CD33 APC (652807), MPO FITC (652821), and CD45 PerCP (652803). Flow cytometry data were collected using Canto‐I (BD Biosciences) cytometers and analyzed with FlowJo V10 software.

Tail bleeds were analyzed on a Hemavet9500 (Drew Scientific). Engraftment was determined from flow cytometry of PB and BM preparations using a FACSCantoII instrument (BD) with analysis by FlowJo software. Our standard flow panel consists of antibodies to block mouse and human Fc IgG receptors (Miltenyi Biotech) as well as mCD45-APC/Cy7(BD), CD45-FITC (BD), CD3-PE/Cy7 (BD), CD19-VioBlue (Miltenyi Biotech), CD13-PE (BD), CD33-PE (BD), CD34-APC (BD), and CD56-v510 (BD). Leukemia percentage was determined by calculating the number of cells with positive staining for CD33 and CD45 (AML) or CD19 and CD45 (ALL) as a fraction of viable cells.

The expression of the myelomonocytic antigens, cluster of differentiation (CD)11b, CD14, CD13 and CD33, on the cell surface was determined by direct immunofluorescence staining and flow cytometry. Briefly, the cells were collected and washed with phosphate-buffered saline (Beyotime). A total of 5×10⁵ cells were stained with the following conjugated antibodies: CD11b-phycoerythrin (PE; catalogue number: 555388), CD14-fluorescein isothiocyanate (FITC; catalogue number: 555397), CD13-PE and CD33-FITC (catalogue numbers 555394 and 555626, respectively, BD Biosciences, Franklin Lakes, NJ, USA). The cells were incubated for 15 min at 4°C and then analyzed using a flow cytometer (FACScabilur; BD Biosciences). Apoptosis assays were performed using an Annexin V-FITC apoptosis detection kit (Beyotime) according the manufacturer’s instructions, and early apoptosis was evaluated by cytofluorometry (FACScabilur, BD Biosciences).