Pe cytm5

Evaluation of post-thaw MSCs generated in culture at each of the 5 sites was conducted by a centralized facility using flow cytometry to determine the expression or lack of expression of surface markers for CD105, CD73, CD90, CD45, CD14, CD34, HLA-DR, and Stro-1. Approximately 250,000 cells were transferred into each of five separate tubes. Cells from one tube were stained with primary antibodies, anti-CD73 conjugated to allophycocyanin (APC) (BD Biosciences), anti-CD90 fluorescein isothiocyanate (FITC) (BD Biosciences) and anti-CD105 phycoerythrin (PE) (Miltenyi Biotec). In a second tube, cells were triple stained with anti-CD45, anti-CD34, and anti-CD14 that were all conjugated with PE-Cy^TM5 (BD Biosciences). In a third tube cells were stained with anti-Stro1 APC (BioLegend) and anti-HLA-DR FITC (BD Biosciences). Additional tubes were stained with isotype controls and one tube was left unstained as a control. All tubes included the use of 7-aminoactinomycin D (7-AAD) to evaluate cells for viability. Following staining, the cells were washed with PBS-BSA, resuspended in PBS-BSA, and were analyzed on a BD FACSCanto (BD Biosciences) flow cytometer.

HCC cells suspended in PBS containing 1% bovine serum albumin were stained with mouse anti-human ITGB1 Ab conjugated with PE-Cytm5 (BD Biosciences, United States) or PE-Cytm5 Mouse IgG1 κ Isotype Control (BD Biosciences, United States) at 4°C for 30 min in the dark. The subpopulation of ITGB1^high and ITGB1^low was obtained using FACSAria SORP cell sorter (BD Biosciences, United States).