Anti cd90 pe cy7

The viability of disaggregated primed and naive hESCs and hiPSCs was analyzed with a Life/Death Fixable kit (Invitrogen, Waltham, MA, USA) in a FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA). Cells were stained with anti-CD24 PE (BD Biosciences, clone ML5) and anti-CD57 APC (BD Bioscience, clone NK-1) primed markers with anti-CD75 FITC (BD Biosciences, clone LN1) and anti-CD130 BV421 (BD Biosciences, clone AM64) naive markers and anti-CD90 PE-Cy7 (BD Biosciences, clone 5E10) stemness marker. Approximately 30,000–50,000 events were acquired for analysis. Populations were analyzed using FlowJo v.X.0.7 (TreeStar Inc., Ashland, OR, USA). See Supplementary Table S1.

The presence of expression markers was assessed by flow cytometry. MSCs were harvested by trypsinisation and labelled with the following antibodies: anti‐CD105‐PE (Southern Biotech; 9811‐09), anti‐CD11b‐PE (BD bioscience; 555388), anti‐CD14‐PE/Cy7 (Biolegend; 301814), anti CD34‐PE (BD bioscience; 345802), anti‐CD45‐PE/Cy7 (BD bioscience; 557748), anti‐CD73‐PE (BD bioscience; 550257), and anti‐CD90‐PE/Cy7 (BD bioscience; 561558). As isotype controls for PE/Cy7 (BD bioscience; 557872) and PE (Beckman Coulter; PN‐AO7796) were used. Cells were labelled in the presence of FcR‐Blocking reagent (Miltenyi Biotec) for 30 minutes at 4°C after which cells were washed twice with FACS‐buffer (PBS, 5% FCS, 0.1% sodium azide). Flow‐cytometric analysis (≥10⁴ events acquired) was performed using a Becton Dickinson FACSCanto II as described (Besseling et al., 2021 (link)).

The presence of EV in SEC fractions was detected using bead-based flow cytometry [55 (link)]. First, 20 μL of the SEC fractions were coupled to 4 μL of 4 μm aldehyde/sulphate-latex beads (Invitrogen-Thermo Fisher Scientific), incubated for 15 min at room temperature (RT), and then blocked in 1 mL BCB buffer (PBS, 0.1% bovine serum albumin (BSA), and 0.01% sodium azide (NaN₃); Sigma Aldrich) for 2 h on rotation. Next, the EV-coated beads were centrifuged at 2000× g for 10 min, and resuspended in BCB buffer. The antibody labelling was performed by incubation (30 min at RT) with the fluorochrome-conjugated antibody anti-CD90-PE-Cy7 (1:50; BD, San Diego, CA, USA) or by indirect labelling with the primary antibodies anti-CD63 (1:10, Clone TEA3/18) and anti-CD9 (1:10, Clone VJ1/20.3.1), followed by incubation with the secondary antibody FITC-conjugated goat F(ab’)2 anti-mouse IgG (1:100; Bionova, Halifax, NS, Canada) or with the Alexa647-conjugated donkey F(ab’)2 anti-mouse IgG (1:100; Jackson ImmunoResearch Europe Ltd., Ely, UK) in CFSE-labelled EV. The beads were washed with the BCB buffer after each incubation by centrifugation at 2000× g for 10 min. Finally, the data were acquired in the FACSLyric flow cytometer (BD) and analyzed using FlowJo v10.2 software (FlowJo, LLC, Ashland, OR, USA).

Third-generation BMSCs were collected and resuspended in complete l-DMEM, and the cell concentration was adjusted to 2.0 × 10⁷ cells/ml. Approximately 50 μl of the cell suspension was placed into the flow tube (Corning, U.S.A.), followed by 5 μl of anti-CD29/AF647, anti-CD90/PE-Cy™7, anti-CD106/PE, anti-CD45/FITC, and anti-CD11b/V450 (BD, U.S.A.). Then, 45 µl of buffer (HyClone, U.S.A.) was added to each tube. The tubes were incubated at room temperature for 30 min, washed twice with staining buffer, and then 500 µl of buffer was added to each tube for detection by flow cytometry (Beckman, U.S.A.).