Anti cd105 fitc

Human CCL5 concentration in the serum of the HCC patients, liver cirrhosis patients and healthy people as well as in the CM of CAFs and PTFs were determined with CCL5 ELISA kit (P13501, RayBiotech) under the guidance of instruction. The following antibodies were used for cytometry: anti-CD105-FITC (800505, Biolegend, San Diego, CA, USA), anti-CD73-APC (344005, Biolegend), anti-CD90-FITC (328170, Biolegend), anti-CD44-FITC (338803, Biolegend), anti-CD34-FITC (343503, Biolegend), anti-CD45-FITC (982316, Biolegend), anti-HLA-DR-FITC (980402, Biolegend) and anti-CD31-APC (303115, Biolegend). The cells staining was performed under manufacturer’s protocol and identified by a flow cytometer (FACSCanto II, BD, USA). FlowJo software (Version X; TreeStar, Ashland, OR, USA) was used to data analysis.

P2 hUC-MSCs were purchased from Cyagen. The cell pellet was cultured in an expansion medium composed of the human umbilical cord mesenchymal stem cell basal medium (HUXUC-01001, Cyagen, China), supplemented with 10% human umbilical cord mesenchymal stem cell cell-qualified fetal bovine serum, 1% glutamine, and 1% penicillin-streptomycin (Cyagen), at 37°C, with 5% carbon dioxide in a fully humidified environment.
Native hUC-MSCs (at P5) were suspended at a concentration of 1 × 10⁶ cells/mL, washed twice in phosphate-buffered saline (PBS), and then incubated for 30 min at 4°C in the dark with the following anti-human antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): anti-CD31-PE (303105), anti-CD45-PE (368509), anti-CD34-PE (343605), anti-CD44-FITC (338803), anti-CD90-FITC (328107), and anti-CD105-FITC (328107; all from BioLegend). After 30 min, cells were washed and resuspended in 300 mL Cell Fix (BD). PE-conjugated IgG1 and FITC-conjugated IgG1 were used as isotype controls (R&D Systems Inc. and Santa Cruz Biotechnology Inc.). Immunophenotyping of hUC-MSCs was performed by flow cytometry.

For immunophenotypic characterization of cells, 1.0 × 10⁶ BMSCs per tube were washed with FACS buffer consisting of PBS supplemented with 1% bovine serum albumin (BSA, Sigma). Then, the cells were incubated for 30 minutes in the dark at room temperature with the following fluorochrome-conjugated primary antibodies: anti-CD90-Percp-Cy5.5, anti-CD73-APC, anti-CD105-FITC, anti-CD146-PE (all from BioLegend, San Diego, CA, USA), anti-CD14-FITC (Immunostep, Salamanca, SPA), anti-CD34-FITC, anti-CD45-Percp-Cy5.5 (both from Agilent DAKO, Santa Clara, CA, USA), and anti-CD11b-PE (Santa Cruz Biotechnology, Dallas, TX, USA). The isotype controls were IgG2A-FITC, IgG1A-APC, IgG1A-Percp-Cy5.5, IgG1-PE, IgG1-FITC, and IgG2A-PE (all from Santa Cruz Biotechnology). Next, the cells were washed with FACS buffer and resuspended in 300 μL buffer for acquisition with a BD FACSCanto™ cytometer (BD Biosciences). The data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).