Pe conjugated anti human cd90

The surface markers of apoVs were identified as in our previous research [16 (link)]. In brief, apoVs were harvested and suspended in Stain buffer and incubated with PE-conjugated anti-human TF (1:50, Biolegend, USA), PE-conjugated anti-human Fas (1:50, Biolegend, USA), PE-conjugated anti-human CD9 (1:50, Biolegend, USA), PE-conjugated anti-human CD63 (1:50, Biolegend, USA), and PE-conjugated anti-human CD81 (1:50, Biolegend, USA), PE-conjugated anti-human CD29 (1:50), PE-conjugated anti-human CD44 (1:50), PE-conjugated anti-human CD90 (1:50), FITC-conjugated anti-human CD34 (1:50), and PE-conjugated anti-human CD45 (1:50) for 30 min at 4 °C. For PS detection, apoVs were suspended in Annexin V Binding Buffer (BD Pharmingen™, USA) and stained with PE-Annexin V (1:50) at 4 °C for 20 min. The positive rate of apoVs were analyzed by NovoExpress™ software.

The uncharacterized cells at passage 3 were seeded in a T-75 cm² flask at a density of 1x10⁶ cells and cultured in the proliferation medium until they reached an 80% confluence rate. Subsequently, cells at the density of 5x10⁵ cells were harvested and had their surfaces analyzed to assess the presence and profile of antigen molecules. This analysis was conducted with the BD FACSMelody™ Cell sorter (BD Biosciences, San Jose, CA, USA). Five specific antibodies were used as markers for MSCs properties, including the APC/Cy7-conjugated anti-human CD73 (1: 100; Biolegend, San Diego, CA, USA), the PE-conjugated anti-human CD90 (1: 100; Biolegend), the Alexa Flour^® 488-conjugated anti-human CD105 (1: 100; Biolegend), and the FITC-conjugated anti-human CD146 (1: 100; Biolegend). The APC-conjugated anti-human CD34 (1: 100; Biolegend), which served as the marker of hematopoietic stem cells, was used as the negative control. The cell pellet was held in 0.1 M PBS-EDTA (Sigma-Aldrich) and used as an unstained compensation. The percentage of the cell-surface antigen molecule markers profiling was counted at 20,000 events and analyzed with the FlowJo™ software (BD Biosciences).

To determine the expression of mesenchymal stem cell markers on T-MSCs, cells were collected and pelleted by centrifugation at 1300 rpm for 5 min. After washing with PBS, cells were pelleted again and stained with FITC-conjugated anti-human CD11b, PE-conjugated anti-human CD34, PerCP-conjugated anti-human CD45, APC-conjugated anti-human CD73, PE-conjugated anti-human CD90, or PE-conjugated anti-human CD105 antibody (Biolegend, San Diego, CA, USA) diluted in FACS buffer (PBS supplemented with 10% FBS, 10 mM EDTA, 20 mM HEPES, and 1 mM sodium pyruvate) for 30 min on ice. To determine the expression of megakaryocytic marker on differentiated K562 cells, Pacific Blue-conjugated anti-human CD41 antibody (Biolegend) was used. After washing cells with buffer, surface protein expression was measured using a NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA) and analyzed using NovoExpress software.