Cd90 pe cy5

The immune phenotype of CIMVs-MSCs was analyzed by immunostaining with monoclonal antibodies: CD90-PE-Cy5 (328112; BioLegend, San Diego, CA, USA), CD29-APC (2115040; Sony, San Jose, CA, USA), CD44-APC/Cy7 (103028; BioLegend, San Diego, CA, USA), CD73-PerCP/Cy5.5 (344014; BioLegend, San Diego, CA, USA). CIMVs were analyzed by flow cytometry (BD FACS Aria III, BD Bioscience, San Jose, CA, USA), and the 405 nm laser was used for better a resolution of CIMVs-MSC.

At passage 3–4, 3 × 10⁵ cells were washed with PBS containing 0,1% sodium azide and 3% FBS and then incubated for 30 min in ice and in the dark with the following mouse anti-human monoclonal antibodies directly conjugated with fluorochromes: CD13/FITC (22A5, Caltag Medsystems, UK), CD14/PE (M5E2, BD Biosciences, CA, USA), CD34/FITC (581, BD Biosciences), CD45/ALexa405 (HI30, Caltag Medsystems), CD90/PECy5 (5E10, BioLegend, CA, USA), CD105/APC (45A5A3, BioLegend), CD146/PE (P1H12, BD Biosciences), CD271/APC (C401457, BD Biosciences). Unstained cells were used as controls. Data was acquired using FACSCanto II (BD Bioscienses) flow cytometer and at least 20.000 events were analyzed with FACSDiva (BD Biosciences) or FlowJo 7.6.5 (FlowJo.com; Tree Star, OR, USA) software.

All UCB-MC units were collected from healthy donors with a gestation period of 37–40 weeks in maternity public hospitals in Kazan. Blood collections were carried out into single blood bags of 250 mL, with the blood preservative CPDA-1 (GCMS, Republic of Korea). Exclusion criteria were maternal infections or viral diseases. Isolations of mononuclear cells were conducted within 16–18 h after blood collection. Nucleated blood cells were isolated using SepMate ™-50 tubes according to the manufacturer’s protocol (STEMCELL Technologies Inc., Vancouver, BC, Canada). The viability of the isolated cells was determined in a hemocytometer with a 0.4% trypan blue solution. Cell viability, as measured by trypan blue exclusion, was >97%. The immune phenotype of isolated cells was analyzed by staining with monoclonal antibodies CD45—PerCP (BioLegend, San Diego, CA, USA), CD3-FITC (BioLegend, San Diego, CA, USA) CD14-APC/Cy7 (BioLegend, San Diego, CA, USA), CD38-APC/Cy7 (BioLegend, San Diego, CA, USA) CD34-FITC (BioLegend, San Diego, CA, USA), CD90-PE/Cy5 (BioLegend, San Diego, CA, USA). Expression of CD markers were analyzed by flow cytometry using BD FACS Aria III (BD bioscience, San Jose, CA, USA)