Cd90 fitc

ADMSC obtained from allogeneic adipose tissue of Sprague–Dawley rats (250–300 g) were cultured. The adipose tissue was digested with collagenase (Sigma Aldrich, Madrid, Spain) and incubated at 37 °C in 5 % CO₂. On the third pass, the cell cultures were divided into three groups: 1) 1.0 × 10⁵ ADMSC for characterization, 2) 1.5 × 10⁶ ADMSC for proteomics analysis of the culture supernatant, and 3) 42 × 10⁶ ADMSC for the treatment of rats. For characterization, ADMSC were trypsinized and labeled with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)- or Alexa 647-conjugated primary antibodies. The cells were incubated for 20 minutes at 4 °C in the dark with the following antibodies: CD90-FITC (AbD Serotec, Oxford, UK), CD29-PE (AbD Serotec), CD45-PE (AbD Serotec) and CD11b-PE (AbD Serotec). Matched isotype controls were purchased from Biolegend (San Diego, CA, USA). Flow cytometry analysis of CD90+/CD29+/CD45–/CD11b– cells was performed using a FACScalibur cytometer and CellQuest Pro software (Becton Dickinson, Madrid, Spain). For ADMSC treatment, ADMSC with >95 % viability were administered i.v. The dose, route and time of administration were based on previously reported data [9 (link), 11 (link)].

Synovial fluid MSCs from three randomly selected donors were characterized by flow cytometry according to the ISCT criteria. The antibodies used were CD90-FITC (AbD Serotec, Kidlington, UK), CD73-PE and CD105-PE (both from BD Biosciences, Wokingham, UK) (MSC positive markers) and CD19-FITC, CD14-PE, CD34-PE, CD45-V450, and HLA-DR-fluorescein isothiocyanate (FITC) (hematopoietic lineage markers) (all from BD Biosciences). A minimum of 10,000 cell events per tube was acquired using Attune flow cytometer (Applied Biosystems). Data were analyzed using FlowJo (BD), and the results are expressed as percentage of positive cells.