Cd11b apc

At 0, 6, 12, and 18 months, PBMCs were collected, and the following immune subsets of cells were analyzed: CD4+ Th, CD8+ Tc, γδ T cells, and OCPs. On Th and Tc cells, we also evaluated the expression of two activation markers, namely, CD69 and CD25. We used the anti-human antibodies CD3 FITC, CD4 APC, PeRCP CD8 PerCP, γδ TCR PE, CD11b APC, CD51-61 PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD25 FITC, CD69 PE (BioLegend, SanDiego, CA, USA), and CD14 FITC (Thermo Fisher Scientific, Waltham, MA, USA), and related isotype and unstained controls. Appropriate controls were used to determine optimal voltage settings and electronic subtraction for the spectral fluorescence overlap correction. Samples were analyzed by the MACsQuant10 instrument and elaborated by MACs quantify software (Miltenyi Biotec, Bergisch Gladbach, Germany). Data represent a percentage of positive cells, determined by subtracting the percentage value of the appropriate isotype and unstained controls from each sample.

Briefly, MSC immunophenotype was established by flow cytometry using the following monoclonal antibodies: anti-CD73-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD90-PE (Miltenyi Biotec), anti-CD105-FITC (Ancell Corporation, Bayport, USA), anti-CD45-PC7 (BD Biosciences), anti-CD34-PE (Miltenyi), anti-CD14-PC7 (BD Biosciences), CD11b-APC (Miltenyi Biotec) and CD19-PC5 (BD Biosciences). After labeling, acquired results were analyzed by using a MACSQuant analyzer (Miltenyi Biotec). Cells were incubated for 30 min with these antibodies and after washing with PBS, the cells were fixed with 8% formaldehyde. BM-MSC were cultured in appropriate induction medium to assess their adipogenic, osteogenic and chondrogenic lineage differentiation capacities (NH media, Miltenyi Biotec). Lipid vacuole formation, mineralization (calcium deposits) and presence of proteoglycans corresponding to each lineage commitment were demonstrated by Oil Red O (Sigma-Aldrich), Alizarin Red S (Sigma-Aldrich) and Alcian Blue (Sigma-Aldrich) staining respectively.

Two different stains were performed with fluorochrome-conjugated antibodies. The first staining consisted of CD45R (B220) PE (Miltenyi Biotech, REA755); CD4 FITC (Miltenyi Biotech, GK1.5); CD8 PerCP-Vio 700 (Miltenyi Biotech, 53–6.7); CD69 APC-Vio 770 (Miltenyi Biotech, H1.2F3); CD44 APC (Miltenyi Biotech, IM7.8.1). The second staining included CD11b APC (Miltenyi Biotech, M1/70); CD103 PE (Miltenyi Biotech, REA789); F4/80 PE-Vio 770 (Miltenyi Biotech, REA126); MHCII APC-Vio 770 (Miltenyi Biotech, REA813). Fc receptors were blocked with Anti-mCD16/CD32 (BD). Only events that appeared single in forward-scatter width were analyzed. FACSCanto II and FACSDiva software (BD) were used for flow cytometry and data were analyzed using FlowJo software (TreeStar).

To analyze the phagocytic capacity of neutrophils derived from DMSO and UM171 expanded hPSC derived HPs, we incubated these neutrophils with opsonized zymosan A fluorescein particles for 1 hour, at 37 °C or 4 °C (control). Samples were subsequently collected on ice, incubated with CD16-PE, CD66b-BV421(BD Pharmingen), and CD11b-APC (Miltenyi Biotech), and counterstained with Ghost violet 540 cell viability dye (TONBO Biosciences). Cells were then analyzed by flow cytometry.

A flow cytometry analysis was performed using a flow cytometer CyFlow (Partec). The cells were washed with FACS buffer (PBS, 2% FBS) and resuspended in a 0.1 mL FACS buffer. The cells were stained with anti-mouse CD105-APC, CD90.2-FITC, CD34-FITC, CD11b-APC (Miltenyi Biotec), CD14 PerCP‐Cy5.5 (eBioscience), Ly-6A/E (Sca-1)-Pacific Blue (BioLegend) antibodies. Data were analyzed using the FlowJo software (BD).

The processed splenocytes were distributed at 1 x 10⁶ per well into Nunc 96-well round bottom microwell plates (Thermo Scientific, UK). The cells were blocked by adding 5μg/ml purified rat anti-mouse CD16/CD32 (mouse BD Fc Block) clone: 2.4G2 (BD Biosciences, UK) in autoMACS buffer (Miltenyi Biotec, UK). Cells were stained with CD8a-FITC (Life Technologies), CD4-PE (Miltenyi Biotec) to detect cytotoxic and helper T cells respectively and B220 biotin RA3-6B2 Alexa Fluor 647 to detect B cells (CD45R), CD11b-APC to detect dendritic cells and CD169 (Siglec-1)-APC to detect marginal zone macrophages. Streptavidin Molecular Probe Alexa-Fluor-633 conjugated secondary mAb (1μg/ml) (Invitrogen) was used to detect biotinylated antibodies 7E9 (BioLegend, UK) and 7G6 (BD Biosciences, UK) to identify CD21/CD35 (CR2/CR1) and 8C12 (BD Biosciences, UK) to identify CD35 (CR1). Single staining controls and no staining controls were also included for compensation purposes. The cells were then fixed with 1% paraformaldehyde, washed and resuspended in MACS buffer, before being read on the MACS Quant (Miltenyi Biotec, UK). The analysis was completed using FCS Express (De Novo Software, US).

For flow cytometry, cells were washed and resuspended in PBS. For cell surface antigen staining, cells were incubated in the dark with fluorescein isothiocyanate (FITC)-conjugated anti-flavocytochrome b558 7D5 (D162-4) (MBL International, Woburn, MA, USA) or CD11b-APC (Miltenyi Biotec no. 130-098-088) antibodies for 20–30 minutes at room temperature. Data acquisition was performed with a BD LSRFortessa flow cytometer (BD Biosciences, Heidelberg, Germany). Data were analyzed with BD FACSDiva software (BD Biosciences, Heidelberg, Germany) or flowing software 2.5.1. Cell sorting was performed in a BD FACSAria III flow cytometer (BD Biosciences, Heidelberg, Germany).