Pe conjugated cd73

FACS was performed using BD FACSAria III (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). To analyze the mesenchymal stem cell markers, 10⁵ cells were stained with antibodies, particularly PE-conjugated CD105 (Bio-Rad, Hercules, CA, USA, MCA1557,), PE-conjugated CD90 (BioLegend, San Diego, CA, USA, 555596), PE-conjugated CD73 (BioLegend, 344004), FITC-conjugated CD45 (BioLegend, 555482), FITC-conjugated CD34 (BioLegend, 343504), and PE-conjugated CD14 (Bio-Rad, MCA1568). To determine the EV-specific marker, isolated EVs were incubated with 4% w/v aldehyde/sulfate-latex beads (Thermo Fisher Scientific, Rockford, IL, U.S, A37304) at room temperature overnight on rotation. Bead-binding extracellular cells were centrifugated at 1800× g for 10 min and washed with 500 µL of PBS. The pellet was resuspended with 50 µL of PBS containing CD63 (BioLegend, 353003) and CD81 (BioLegend, 349505) at 4 °C for 1 h. All antibodies were conjugated with PE fluorescence dye. Samples were washed with 500 µL of PBS and centrifuged at 1800× g for 10 min. The pellet was resuspended with PBS. Gating of exosome-decorated beads approximately 4 µm in diameter was analyzed using BD FACSAria III (BD Biosciences, San Jose, CA, USA). At least 10,000 events were acquired on flow cytometry. Data analyses was performed using BD FACSDiva™ software version 6.1.3 (BD Biosciences, San Jose, CA, USA).

Cells were dissociated into single cells with 0.05% trypsin (0.1% EDTA) wherever suitable or rinsed off from culture, and resuspended in a FACS washing buffer (PBS with 5% fetal calf serum (FCS) and 2.5 mM EDTA). The cell suspension was then stained with the desired antibodies. The antibodies used in this study were: CD56 (1:50, clone CMSSB; eBioscience), PE-conjugated CD309 (FLK1, 1:50, clone 7D4–6; Biolegend), PE-conjugated CD13(1:50, clone WM15; BD), FITC-conjugated CD31 (1:50, clone WM59; BD), APC-conjugated CD34 (1:50, clone 581; BD), FITC-conjugated CD43 (1:50, clone MEM-59; Biolegend), PE-conjugated CD43 (1:20, clone eBio84-3C1; eBioscience), FITC-conjugated CD45(1:50, clone 5B1; Miltenyi Biotec), FITC-conjugated CD14 (1:50, clone HCD14; Biolegend), PE-conjugated CD68 (1:50, clone Y1/82A; Biolegend), APC-conjugated CD11b (1:50, clone ICRF44; Biolegend), PE-conjugated CD163 (1:50, clone RM3/1; Biolegend), PE-conjugated CD73 (1:50, clone AD2; Biolegend) and APC/Cy7-conjugated CD163 (1:50, clone 12G5; Biolegend). FITC-conjugated mouse IgG2a (1:20, 130-098-846; Miltenyi Biotec), APC-conjugated mouse IgG1 (1:20, 130-098-877; Miltenyi Biotec) and PE-conjugated mouse IgG1κ (1:20, clone P3.6.2.8.1; eBioscience) were used as isotype-matched negative controls. Data were collected with a FACS Calibur flow cytometer (BD) and analyzed using FlowJo software, version 10.0.7.

The immunophenotype was analyzed within the 4th and 5th passages by fluorescence-activated cell sorting (FACS). In brief, 5 × 10⁵ cells (in 50 μl of staining buffer (SB, Pharmingen Becton Dickinson)) were mixed with 10 μl of the following antibodies: PE-conjugated CD73 (BioLegend, San Diego, CA), FITC-conjugated CD90 (Santa Cruz Biotechnology, California), PE-conjugated CD34 (BD Biosciences, NJ), RPE-conjugated CD45 (BD Biosciences, NJ), RPE-conjugated CD14 (Serotec, Kidlington, UK), and APC-conjugated CD105 (BioLegend). Proper isotype controls for each antibody were used to discard unspecific binding. Cells were incubated for 20 min at room temperature in the dark and then fixed and analyzed using an Attune flow cytometer (Life Technologies, Carlsbad, CA). Data analysis was performed using FlowJo version 7.2.2 software (Tree Star Inc., Ashland, OR). Nonviable cells were excluded according to the side scatter vs. forward scatter parameters, and 5,000 events were acquired for each sample.