Fitc conjugated mouse anti human cd90

The ECFCs were mixed with BM-MSCs at a ratio of 2:1 in a rat tail collagen type 1-coated T-25 flask for an initial period of 4 h in EGM-2, and later changed to EBM-2 for a further duration of 48 h. Monocellular cultures were used as controls. Pure ECFCs were then sorted using CD31 microbeads (130-091-935, Miltenyi Biotec, San Diego, CA, USA) via magnetic-activated cell sorting. The sorted ECFCs were further maintained at general culture conditions, and will be referred to as directly primed ECFCs. To ensure that directly primed ECFCs were free from MSC contamination, harvested cells were further labeled with 1.0 µL of FITC-conjugated mouse anti-human CD31 (Cat. 303,103; BioLegend, San Diego, CA, USA) and 1.0 µL of FITC-conjugated mouse anti-human CD90 (Cat. FAB357P; R&D Systems, Minneapolis, MN, USA) at 4 °C for 30 min, and stained cells were detected using an LSR Fortessa X-20 (Becton Dickinson, Franklin Lakes, NJ, USA). ECFCs were considered to be positive for the expression of CD31, but negative for the expression of CD90.

Type-I collagenase, and type-I hyaluronidase were purchased from Sigma-Aldrich (St Louis, MO, USA). FACS lysing solution and Cytofix/Cytoperm solution were obtained from BD Pharmingen (San Jose, CA, USA). A rabbit anti-human LRG1 antibody, a FITC-conjugated mouse anti-rabbit IgG antibody, and a PE-conjugated mouse anti-rabbit IgG antibody were purchased from LifeSpan BioSciences (Seattle, WA, USA). PerCP-conjugated mouse anti-human CD4, PE/Cy7-conjugated mouse anti-human CD8, APC/CY7-conjugated mouse anti-human CD14, APC-conjugated mouse anti-human CD19, APC/Cy7-conjugated mouse anti-human CD16, PE/Cy7-conjugated mouse anti-human CD123, PerCP-conjugated mouse anti-human HLA-DR, PE/Cy7-conjugated mouse anti-human CD34, PerCP-conjugated mouse anti-human FcεR1, PE-conjugated mouse anti-human CD117, FITC-conjugated mouse anti-human CD90, APC-conjugated mouse anti-human TGFBR2, and PE-conjugated mouse anti-human TGFBR2 antibodies were purchased from BioLegend (San Diego, CA, USA). Allergens for skin prick tests were supplied by ALK-Abelló, Inc. (Denmark). Human LRG1, TGFBR2, and tryptase ELISA kits were purchased from R&D Systems (Minneapolis, MN). Most general-purpose chemicals such as salts and buffer components were of analytical grade.