Cd61 pe

Each assay contained a unique combination of the following mouse anti-human monoclonal antibodies: CD73-FITC, CD90-APC, CD105-PE (BioLegend, San Diego, CA, USA), CD34-PE, CD36-APC, CD146-PE (Miltenyi BioTech, Bergisch, Germany), CD61-PE (Beckman Coulter Inc., Pasadena, CA, USA), CD15-FITC, and 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA). All antibodies were titrated and used at a 50 ng/assay concentration. Isotype controls and specific mAbs were employed at the same final concentrations. Routinely, 50,000 hASCs (in 100 µL FACS buffer) were mixed with the appropriated antibody combination and incubated for 15 min at RT in the dark. Finally, the sample was diluted with 100 µL FACS buffer before the acquisition, according to our flow cytometer procedure (see Section 2.3.3).

Platelets were gated using CD42b-APC and CD61-PE (#551061, #IM3605; Beckman Coulter) antibodies. For the isolation of leukocytes, whole blood was drawn into Vacutainer EDTA tubes. Erythrocytes were lysed using a TQ-Prep Workstation (Beckman Coulter) and the remaining cells were washed with PBS. Lymphocytes and monocytes were gated using CD5-PC5.5 and CD14-PC7 antibodies (#B49191, #A22331; Beckman Coulter), respectively. After exclusion of lymphocytes and monocytes, granulocytes were gated by forward (FSC) and side scatter (SSC) characteristics. α-2,6- and α-2,3-linked sialic acid was detected using 250 ng/mL Cy5-labeled Sambucus nigra lectin (SNA) (#CL-1305; Vector Laboratories) and 100 μg/mL FITC-labeled Maackia amurensis lectin II (MAL II) (#21511103-1; GlycoMatrix), respectively. Cells were acquired on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed with FlowJo software (Tree Star). To adjust for distinct autofluorescent properties of investigated cells, results were presented as delta geometric mean fluorescence intensity (Δ gMFI) in relation to buffer treated cells.