Cd45 fitc

Flow cytometric analysis of chondrocyte sheets was performed on single-layer sheets before layering and on triple-layered sheets on the day before transplantation. Chondrocyte sheets were digested with TrypLE Express (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 15 min and then incubated with 0.25 mg/ml collagenase-P (Roche, Basel, Switzerland) at 37 °C for 30 min. The dispersed cells were washed with Ca⁺⁺/Mg⁺⁺-free phosphate-buffered saline (PBS) containing 0.2% human serum albumin (Sigma-Aldrich, St. Louis, MO, USA) and 1 mM ethylenediaminetetraacetic acid (EDTA; Wako Pure Chemical Industries, Ltd.), and then immunostained with the following antibodies: CD31–fluorescein isothiocyanate (FITC) (clone: 5.6E) and CD45–FITC (clone: J.33) from Beckman Coulter, Inc. (La Brea, CA, USA); CD81–allophycocyanin (APC) (clone: JS-81), CD90–APC (clone: 5E10), CD49a–phycoerythrin (PE) (clone: SR84), and disialoganglioside GD2 (clone: 14.G2a) from BD Biosciences; and CD146–PE (clone: F4-35H7 (S-Endo 1)) from BioCytex (Marseille, France). Fluorochrome-labeled mouse IgG1 antibody (clone: 679.1Mc7, Beckman Coulter) was used as a negative control and FITC-conjugated goat anti-mouse IgG (BD Biosciences) was used as the secondary antibody. Stained cells were analyzed using a FACSVantage flow cytometer (BD Biosciences).

Phenotypic analysis of MSC was performed during monolayer expansion (prior to encapsulation in the hydrogel) and throughout scaffold culture. Briefly, to perform phenotypic analysis MSC were incubated with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mouse anti-human antibodies CD34-PE, CD45-FITC, HLA-DR-FITC, CD44-FITC, CD73-PE, CD90-FITC, CD105-PE and CD166-PE (Beckman Coulter, Brea, CA, USA) for 30 minutes at room temperature. Negative and isotype (FITC and PE) controls were performed. After immunofluorescence staining, for each sample 10,000 events were counted by Gallios flow cytometer (Beckman Coulter, Brea, CA, USA).

Blood samples were collected at W0, W8, W24 and W48 for immunophenotyping by flow cytometry in ethylenediaminetetraacetic acid (8.55 mg/tube). Peripheral blood samples (100 µL) were incubated with specific monoclonal antibodies (Beckman Coulter, Wycombe, UK) at room temperature (RT) for 15 min in the dark; afterward, the red cells were lysed, at RT in the dark, in a VersaLyse lysing solution (A09777, Beckman Coulter, Wycombe, UK) for 15 min and then analysed. LS were identified by the recognition of surface molecules belonging to the CD family. The samples successively underwent gating analysis (total lymphocytes and CD45+ vs. side scatter) (CD45-FITC, A07782, Beckman Coulter, Wycombe, UK), and the desired lymphocyte subpopulations were gated excluding doublets. The LS identification was based on monoclonal antibodies against Bmem CD19+/CD27+, Br1 CD19+/CD38+, Br2 CD19+/CD25+, Treg CD4+/CD25+, Th CD3+/CD4+ and Tc CD3+/CD8+ (CD19-PC7, IM3628; CD27-PE, IM2578; CD38-PE, AO7779; CD25-PE, A07774; CD4-FITC, A07750; CD3-PC5, A07749; CD8-PE, A07757; Beckman Coulter, UK). The samples were tested using a CytoFLEX flow cytometer and the CytExpert software (Beckman Coulter, Wycombe, UK).