Cd45 pe

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)].

hUC-MSC-p3 and hUC-MSC-p15 were collected and treated with 0.25% trypsin. The cells were individually stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated anti-marker monoclonal antibodies in 100 μl PBS for 15 min at room temperature, or for 30 min at 4°C, as recommended by the manufacturer. The antibodies used were specific for the following human antigens: CD34-PE, CD44-FITC, CD45-PE, CD73-PE, CD90-PE and CD105-PE (10 μl for 1×10⁶ cells; AbD Serotec, Raleigh, NC, USA). Cells were analyzed on a Cytometer 1.0, Cytomics™ FC500 flow cytometry system (Beckman Coulter, Brea, CA, USA). Positive cells were counted and the signals for the corresponding immunoglobulin isotypes were compared (15 (link)).

The hAD-MSCs were provided by Cellerix and were obtained from lipoaspirates from healthy donors. The rAD-MSCs were extracted from abdominal adipose tissue of adult Sprague–Dawley rats (250 to 300 g) as previously described [2 (link)]. Briefly, the extracted tissue was washed with sterile PBS and digested with an equal volume of 0.075% type I collagenase (Sigma-Aldrich). The filtered cells were centrifuged at 390g for 10min and contaminating erythrocytes were removed to isolate the stromal vascular fraction (SVF) that was cultured in Dulbecco modified Eagle medium (1X) (DMEM Glu/Pyr, Gibco), 75 μl penicillin/streptomycin (Sigma-Aldrich) and 20% fetal bovine serum (PAA Laboratories). On the third pass, cells were trypsinized and counted before being administered to the experimental animals.
To confirm the presence or absence of MSCs surface markers using the flow cytometric technique, the cells were incubated for 20 min at 4°C in the dark with the following antibodies: CD90-fluorescein isothiocyanate (FITC) (AbD Serotec), CD29-Phycoerythrin (PE) (AbD Serotec), CD45-PE (AbD Serotec) and CD11b-PE (AbD Serotec). Matched isotype controls were purchased from Biolegend. At least 1×10⁴ cells per sample were acquired and analyzed [2 (link)].