Cd11b pe

Cells were treated with FcR blocking reagent (Miltenyi Biotec, Germany) and stained with primary antibodies CD45-FITC (Becton, Dickinson and Company, USA), CD11b-PE and CD14-APC (Beckman Coulter, USA) at dilutions of 1:25. For negative controls, primary antibodies were replaced with mouse IgG1 (BD). DAPI (Sigma-Aldrich) was used to exclude dead cells. The flow cytometric analysis data were collected using a MACSQuant Analyzer (Miltenyi Biotec) and analyzed using the FlowJo software program (TreeStar, USA).

Annexin-APC and PI (Thermo Fisher Scientific) were used for apoptosis analysis. CD11b-PE (Beckman Coulter, Tokyo, Japan) was used for granulocytic differentiation analysis. CD14-Alexa Fluor (Thermo Fisher Scientific) was used for monocytic cell differentiation. Cells were incubated for 15 min with indicated antibodies and then detected using LRSII (BD Biosciences, San Jose, CA). Results were analyzed using the FlowJo software (version 9.3.2).

2.5 × 10⁵ of PBMCs were suspended in 50 μL of phosphate-buffered saline (PBS) and incubated in the dark for 15 min at room temperature with 20 μL of HLA-DR_PerCP, CD11b_PE, and CD14_FITC antibodies (Becton Dickinson, CA, USA). Then, the cells were resuspended in 500 μL of PBS. The monocytes were detected by a three-color flow cytofluorimeter (Beckman Coulter, CA, USA) with positive controls for CD11b_PE and CD14_FITC. Monocyte HLA-DR measurements were expressed as percentages of HLA-DR-positive monocytes and as means of fluorescence intensities (MFI) in relation to the entire monocyte population, thus reflecting the HLA-DR density per cell. Flow cytometry analysis was performed using Kaluza software V1.1 (Beckman Coulter, CA, USA). Setting gates were based on the internal negative population. The figure of analysis strategy for monocyte HLA-DR expression was presented in our previously published paper [13 (link)].

The characterization of the LSC population and its differentiation was evaluated by direct immunofluorescence staining using different combination of the following monoclonal antibodies: CD34-FITC, CD34-PE, CD38-PECy5, CD123-PE, CD11b-PE (Beckman Coulter). Mouse IgG antibodies were used as isotype controls.
MRP4/ABCC4 protein expression was detected by indirect immunofluorescence staining using the affinity purify polyclonal anti-human MRP4 antibody (1/50) [46 (link)]. The secondary antibody used in 1/200 dilution was goat anti-rabbit AlexaFluor 488 (Invitrogen). Incubations with antibodies were performed in PBS 2% FCS for 45 min at 4°C. Cells were washed twice with PBS 4% FCS and resuspended in PBS. Incubation without MRP4 antibody was performed as a negative control.
Data were acquired using a CyAn ADP Flow Cytometer (DakoCytomation, Beckman Coulter) and analysed using FlowJo software (Treestar).

CIKs and MSCs were harvested, transferred into PBS, and incubated with Fc-solution (Beckman Coulter) for 5 min at 4°C to prevent unspecific antibody binding. During the incubation with Fc-blocking reagent, antibodies were placed in fluorescence-activated cell sorter (FACS) tubes (Greiner, BIO). The following antibodies, purchased from Beckman Coulter (final concentrations in brackets), were used: CD3-FITC (1∶100), CD4-PE (1∶250), CD8a-PE (1∶250), CD11b-PE (1∶160), CD19-PE (1∶250), CD25-PE (1∶250), CD45-PE (1∶250), CD49-PE (1∶250), NK1.1-PE (1∶60), CD69-PE (1∶250), HamIgG-FITC-isotype-control (1∶250), and RatIgG2a-PE-isotype-controll (1∶250). In addition, CD34-PE (Caltaq, 1∶100), CD44-AF488 (Biozol, 1∶250), CD73-PE (Biozol, 1∶100), CD90 (Thy1.2)-FITC (Miltenyi, 1∶100), and CD166 (Antikörper-online, 1∶100).
After incubation for 5 min, Fc-blocked cells were added to the antibody-containing FACS tubes, incubated for 20 min at 4°C, and washed with PBS. Then, 7-aminoactinomycin-D (7-AAD; 1∶100, Beckman Coulter) stain was added, and the cells were incubated for 5 min at 4°C. Either isotypes or autofluorescence served as controls. Cells were gated on vital cells, indicated by low 7-AAD signals.

Apoptotic cells were detected with Annexin V-FITC detection kit from BD Pharmingen (San Diego, CA, USA) according to the manufacturer instructions. To evaluate AML cell differentiation, cells were stained for 30 min with the following anti-human monoclonal antibodies: CD11b-PE (Beckman Coulter), CD14-FITC (BD Biosciences), CD15-APC (BD Biosciences). For patient samples, additional hCD45-APC-H7 (BD Biosciences), Annexin V-Pacific Blue (BioLegend, San Diego, CA, USA) and 7-AAD (Sigma) were used. Data were collected on a LSRII or a LSRFortessa cytometer (BD Biosciences), and analyzed with FlowJo software. A minimum of 10,000 events was collected.