Cd14 apc

Whole human blood was incubated in ACK RBC lysis buffer (Thermo Fisher Scientific) for 4 min at 37 °C to remove red blood cells. Cells were incubated with NIR Zombie (1:200, Biolegend) in PBS for live/dead staining for 15 min at room temperature, followed by BV421 CD45 (1:200, clone 2D1, #368522, Biolegend), APC CD14 (1:200, clone 63D3, #367118, Biolegend), BV605 CD16b (1:200, clone CLB-gran 11.5, #735143 BD), PE/Cy7 CD19 (1:200, clone HIB19, #302216, Biolegend), PE/Cy7 CD3 (1:200, clone HIT3a, #300316, Biolegend) and Fc blocker (1:100, anti-mouse CD16/32,BD) at 4 °C in the dark for 20 min, and washed in PBS/0.5% BSA. Neutrophils, lymphocytes, and monocytes were identified from peripheral blood cells of normal healthy donors with the following high dimensional immunophenotyping; Neutrophils (CD45⁺, CD3/CD19−, CD16b+, CD14⁻), lymphocytes (CD45⁺, CD3/CD19+, CD16b−, CD14⁻) and monocytes (CD45⁺, CD3/CD19−, CD16b−, CD14⁻). Analysis was performed using an LSRII (BD) flow cytometer and FlowJo. v10 (Treestar).

For cell fusion analysis, CD14⁺ monocytes were pretreated with 100 ng/ml of RANKL and 50 ng/ml of M-CSF for 3 days. Then cells were surface stained with APC-CD14, FITC-CD36, FITC-DC-STAMP, and FITC-CD47 mouse polyclonal antibodies (BioLegend, San Diego, CA, USA).

For cell phenotype analysis, monoclonal antibodies were directly added to 100 μL aliquots of heparinized whole blood and incubated at room temperature for 15 min. The antibodies used were: CD3 PECy7, CD14 APC, CD56 PE-Cy5 and CD19 PerCp (Biolegend, San Diego, CA, USA), CD4 Viogreen (Miltenyi Biotec GmBh, Bergisch Gladbach, Germany), CD39 Fitc, CD73 PE, CD25 BV421 and CD127 AlexaFluor 647 (BD Biosciences, San Jose, CA, USA). Red blood cells were lysed and white cells were fixed using BD FACS lysing solution (BD Bioscience). Negative populations were determined using respective anti-human isotype controls. Samples were acquired using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec GmBh) and the data were analyzed using FlowJo software v. 10.0 (TreeStar Inc., Ashland, OR, USA).
We compared the frequencies of four subsets based on CD39 and CD73 expression (CD39⁻CD73⁺, CD39⁺CD73⁺, CD39⁺CD73⁻, and CD39⁻CD73⁻) within each studied population (B cells as CD3⁻CD19⁺, CD4⁺ T cells as CD3⁺CD4⁺, CD8⁺ T cells as CD3⁺CD4⁻, NK cells as CD3⁻CD4⁻CD19⁻CD56⁺, Tregs as CD3⁺CD4⁺CD25⁺⁺CD127⁻, and monocytes as CD14⁺). The percentage of these four subsets was related to their corresponding precursor populations (CD4⁺ T, CD8⁺ T, B, NK, and monocytes). In the case of Treg, the percentage was related to CD4⁺ T cells.

Single cell suspensions obtained from colony forming assays or hematopoietic organs from mice were surface stained with monoclonal antibodies. Antibodies used for analysis of human immature stem and progenitor cells from colony forming assays: CD34 PE-Cy7(581), CD38 APC (HIT2), CD10 PE/PeCy5/APC (HI10a), CD45RA PerCP-Cy5.5 (HI100), CD90 APC-Cy7 (5E10) (Biolegend). Antibodies for erythroid cells from colony assays and HEMA: CD117 PE-Cy7 (104D2), CD71 APC/APC-Cy7 (CY1G4) (Biolegend), CD235a BV421 (HIR2) (BD biosciences). Antibodies for myeloid cells from colony forming assays: CD34 PE-Cy7 (581), CD33 PE/PerCP-Cy5.5 (WM53), CD14 APC (M5E2), CD115 BV421 (g-4D2-1E4), CD15 PeCy5 (W6D3), CD66b PerCP-Cy5.5 (G10F5) (Biolegend). Antibodies for megakaryocyte populations: CD41 PerCP-Cy5.5 (HIP8), CD61 APC (VI-PL2) (Biolegend). Antibodies for overall human and human myeloid cells in xenograft assay: CD45 Biotin (HI30), CD34 PE-Cy7/APC-Cy7 (581), CD33 PE/PerCP-Cy5.5 (WM53), CD15 PeCy5 (W6D3). Antibodies for human B cells in xenograft assay: CD19 PE-Cy7 (HIB19), IgM APC-Cy7 (MHM-88), CD10 APC (HI10a) (Biolegend). Antibody to detect murine cells in xenograft assay: CD45 PE-Cy7/PerCP-Cy5.5 (30-F11) (Biolegend). Streptavidin APC/V450 (Biolegend) were used as secondary antibodies. BD LSRFortessa was used for flow cytometry. Analyses were performed using FlowJo.

Thawed PBMC were washed twice with complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l‐glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 μM β‐mercaptoethanol) plus 0.02 mg/ml DNAse. PBMCs were stimulated for 4 h at 37°C in a 5% CO2 atmosphere with PMA (100 ng/ml) and Ionomycin (1 μg/ml) in complete culture medium. For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson). After stimulation, cells were stained with LIVE‐DEAD Aqua (Thermo Fisher Scientific) and surface mAbs recognizing HLA‐DR‐PE‐Cy7, CD14‐APC, and CD16‐BV421 (BioLegend, San Diego, CA, USA). Cells were washed with stain buffer, fixed, and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Then, cells were stained with previously titrated mAbs recognizing IFN‐γ‐FITC and TNF‐BV605 (all mAbs from BioLegend). Samples were acquired on Attune NxT acoustic cytometer (Thermo Fisher).

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.