Cd45 apc cy7

BM cells were incubated with Fc Block (BD Bioscience) for 15 min, and then stained with the following conjugated antibodies (30 min at 4°C in the dark): CD45-APC-Cy7, NK1.1-APC, CD3-APC, CD19-APC, CD11b-PerCP-Cy5.5, Ly6C-FITC (all from BD Bioscience), TER119-APC, Ly6G-eFluor450 (eBioscience) for neutrophil and monocyte populations, CD45-APC-Cy7, CD19-PE, CD4-V500, CD8-PerCP-Cy5.5, Gr1-APC (BD Bioscience), and CD3-eFlour450, NK1.1-PE-Cy7, TER119-APC (eBioscience) for lymphocyte population. After antibody staining, the cells were incubated with 7-AAD for 5 min at room temperature as a viability dye for dead cell exclusion and analyzed in the presence of AccuCheck counting beads by FACSSymphony A3. The data were further analyzed by using the FlowJo software.

To assess the capacity of MSCs to induce the generation of T-regulatory (Treg) cells, PBMC-MSC cocultures have been set up. In short, 500 × 10³ PBMCs from five different donors obtained by Ficoll gradient centrifugation were plated in the presence of 50,000 allogeneic MSCs in RPMI 10% FBS with IL-2300 U/ml in a 24-multiwell flat-bottomed plate for 1 week of culture [36 (link)]. At day 7, PBMCs were harvested and analyzed by flow cytometry after incubation with the following conjugated monoclonal antibodies: CD25 FITC, CD127 PE, CD4 APC, CD3 PECy7 and CD45 APCCy7 (Becton Dickinson). Treg cell induction by MSCs was evaluated as the percentage of CD25^High/CD4⁺/CD127^Low/− PBMCs [37 (link)].

Immune cells were isolated from mouse liver, as described previously.¹ (link)^,27 (link) Immune cells were stained with CD45-APC-Cy7 (Becton Dickinson, Franklin Lakes, NJ), CD11b-PE (Becton Dickinson), F4/80-FITC (Miltenyi Biotec), and Ly6C-Pacific blue (MACS). Flow cytometry was carried out using BD LSR-Fortessa. Analysis was performed using FlowJo software (FlowJo 10.8.1, Ashland, OR).

Freshly BM and PB samples were stained with suitable conjugated antibody: PE-LIGHT (R&D Systems), CD45-APC-Cy7, FITC-CD8, FITC-CD4, Pe-Cy-5-CD14 and FITC-CD16 (Becton Dickinson, Milan, Italy). Flow cytometry analysis was performed on a FACSCantoTM II or BD Accuri™ C6 flow cytometer (Becton Dickinson Immunocytometry System, Mountain View, CA, USA). Positivity area was determined using an isotype-matched mAb, and a total of 2000 events for each cell sub-population was acquired.

Generation of single-cell suspension was performed as described in Llorens-Bobadilla et al.⁶ (link) Cells were stained with the following antibodies: O4-allophycocyanin (APC) and O4-APC-Vio770 (Miltenyi; diluted 1:50), Ter119-APC-Cy7 (BioLegend; 1:100), CD45-APC-Cy7 (Becton Dickinson [BD]; 1:200), GLAST (ACSA-1)-phycoerythrin (PE; Miltenyi: 1:20), CD9-eFluor450 (eBioscience; 1:300), Alexa647::EGF (Life Technologies; 1:100), polysialylated neuronal cell adhesion molecule (PSA-NCAM)-PE-Vio770 (Miltenyi; 1:75), Prominin1- peridinin-chlorophyll-protein PerCP-eFluor 710 (eBioscience; 1:75), CD24-PE-Cy7 (eBioscience; 1:75), and Sytox Blue (Life Technologies; 1:1,000). For RNA-seq, cells were directly sorted into 100 μL of the PicoPure RNA Isolation Kit (Thermo Fisher Scientific) extraction buffer. For ex vivo transduction, NSCs were sorted into growth factor-free Neurobasal medium (NBM).

For surface staining, single-cell suspensions prepared from the spleens of hu-mice or total blood cells collected in EDTA-coated tubes were stained for surface markers and analyzed on a BD LSRFortessa Beckton-Dickinson). Human CD11c-PacBlue, CD45-AF700, CD45-APC-Cy7, HLA-DR-PeCy7, CD40-PeCF, CD19-PeCy5, CD19-APC, CD19-PacBlue, CD27-APC, IgG-PeCy5, IgG-PercpCy5.5, IgD-FITC, IgM-FITC, CD3-APC-H7, CD3-FITC, CD14-FITC, CXCR5-AF488, and streptavidin-PE were purchased from Becton-Dickinson, whereas human CCR7-PacBlue, CD45RA-FITC, IgM-PacBlue, and ICOS-APC were purchased from Biolegend. Pacific orange-conjugated anti-mouse CD45, PE/Texas red-conjugated anti-human CD4, and the LIVE/DEAD Fixable Aqua (LD7) Dead Cell Stain Kit were purchased from Invitrogen. Data were analyzed using FlowJo software (FlowJo, LLC).

In order to assess the immunomodulatory potential of MSCs in both culture conditions, a mixed leukocyte reaction (MLR) was performed. Peripheral blood mononuclear cells (PBMCs) from five different donors were obtained by Ficoll (Lymphosep, Lymphocyte Separation Media; BioWest) gradient centrifugation and resuspended in RPMI1640 medium (Euroclone) supplemented with 10% FBS.
Stimulator mononuclear cells (S) were irradiated at 60 Gy before being cultured with responder PBMCs (R). Then 200 × 10³ responders and 100 × 10³ stimulators were mixed with different concentrations of MSCs to obtain different concentration ratios (1:5, 1:10, 1:20, 1:100) in flat-bottomed 96-well plates (Corning) for 6 days. As controls, a polyclonal stimulation with purified anti-CD3/28 antibodies (BD Pharmingen) was also performed on PBMC responders in the presence of MSCs. Proliferation was measured on day 6 by carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (Vybrant CFDA SE Cell Tracer Kit; Thermofisher) labeling of responder PBMCs. A FACS analysis on flow cytometry was performed on responder PBMCs after incubation with the following conjugated monoclonal antibodies: CD25 PE, CD45 APCCy7, CD3 APC and HLA-DR PECy7 (Becton Dickinson). The immunomodulatory effect is evaluated as a percentage of proliferation (% CFDA-SE-positive cells) in the presence/absence of MSCs on 7-AAD⁻ CD45/CD3⁺ cells.