Cd44 apc cy7

The immune phenotype of EVs was analyzed using flow cytometry (BD FACS Aria III, BD Bioscience, East Rutherford, NJ, USA) by immunostaining with the CD49e-PE (Sony, Tokyo, Japan), CD63-FITC (Biolegend, San Diego, CA, USA), Sca1-APC/Cy7 (BioLegend), CD45-PE/Cy7 (BioLegend), CD9-APC (Biolegend) and CD44-APC/Cy7 (BioLegend) antibodies.

Lymphoid cells from bone marrow, thymus and spleen were isolated from 8-week old mice. To reveal T cell progenitor subsets in the thymus, single cell suspensions were stained with conjugated antibodies, specific for CD3-FITC (Biolegend), CD4-APC, CD8a-PerCp-Cy5.5, CD25-PE, CD44-APCCy7 (Biolegend), TCRβ-Pacific Blue (Biolegend). To analyse lymphoid subsets in the spleen, single cell suspensions were stained with CD3-FITC, CD4-APC, CD8a-PerCp-Cy5.5, CD19-APCH7, CD45R(B220)-PacificBlue, IgD-PE (eBioscience), IgM-PECy7 (eBioscience). To define the B cell progenitor subsets, suspensions from the bone marrow were stained with IgD-FITC, CD25-PE, IgM-PECy7 (eBioscience), CD45R (B220)-PacificBlue, CD117 (cKit)-APC (eBioscience), CD19-APCH7. Dead cells were excluded from the analysis by propidium iodide staining. Antibodies were purchased from BD Pharmingen unless mentioned otherwise. Samples were measured on a FACS Fortessa^® and analysed using FlowJo^® software (Version: 10.0.8r1).

Flow cytometry was performed with a LSRII machine (Becton Dickinson) to analyze T-cell developmental and cell death markers from healthy Lck-Dlx5 mice. CD4-APC/Cy7, CD8-PE, CD44-APC/Cy7, CD25-PE, Notch1-Alexa647 and Notch3-APC antibodies were obtained from BioLegend. Annexin V-FITC and ethidium homodimer III were from Biotium. Data were analyzed with FlowJo software.

Collagenase type I, penicillin/streptomycin (Pen/Strep) broad-spectrum antibiotic cocktail, trypsin-EDTA (0.25%), fetal bovine serum (FBS), human insulin and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Gibco/Invitrogen (Carlsbad, CA, USA). VersaLyse^TM was purchased from Beckman Coulter (Miami, FL, USA). Dexamethasone, 3-isobutyl-methylxanthine, Nile Red (NR) and indomethacin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vybrant® DyeCycle^TM Violet was purchased from Thermo Fisher Scientific/Life Technologies (Eugene, OR, USA). The following mouse anti-human monoclonal antibodies were purchased from Biolegend (San Diego, CA, USA): CD14-APC Cy7 (Clone M5E2), CD31-PE Cy7 (Clone WM-59), CD36-APC (Clone 5-271), CD73-FITC (Clone AD2), CD44-APC Cy7 (Clone IM7) and CD105-PE (Clone 42A3). Mouse anti-human CD45-Krome Orange (Clone J.33), CD90-PE-Cy5 (Clone Thy-1), CD34-PE Cy7 (Clone 581), and the viability dye, 7-aminoactinomycin D (7-AAD) were purchased from Immunotech/Beckman Coulter (Marseille, France).

MSCs were isolated from subcutaneous adipose tissue of the adult mice (8–9 weeks old). Adipose tissue were dissected into small pieces and digested with 0.2% collagenase II (Dia-M, Russia) in a shaker-incubator at 37 °C, 120 rpm for one hour. Suspended cells were pelleted (500 g for 5 min), washed once in PBS (PanEco, Russia) and re-suspended in DMEM (PanEco, Russia) supplemented with 10% fetal bovine serum (Gibco, UK) and 2 mM l-glutamine (PanEco, Russia). MSCs were maintained at 37 °C, 5% CO₂ with culture medium being replaced every 3 days. MSCs from passages 3 and 4 were used for the experiments.
MSCs were differentiated into three lineages: adipogenic, chondrogenic and osteogenic and it was confirmed by detection of lipid droplets (Oil Red dye staining), glycosaminoglycans and mucins (1% alcian blue staining) and calcium deposits (5% AgNO₃ staining), respectively. Immune phenotype was determined using monoclonal antibodies to CD90.2-PerCP (1301575; Sony, USA), CD44-APC/Cy7 (103028; BioLegend, USA), CD73-Alexa Fluor647 (127208; BioLegend, USA), CD49e-PE (1119525; Sony, USA), Sca1-APC/Cy7(108126; BioLegend, USA), CD29-PE (102208; BioLegend, USA), CD11b-PE/Cy7 (101216; BioLegend, USA), CD10-PE (312203; BioLegend, USA), CD45-PE/Cy7 (103114; BioLegend, USA). Expression of CD markers was analyzed by flow cytometry (BD FACS Aria III (BD Bioscience, USA)).