Fitc mouse anti human cd90

To study the transfer of Poly(I:C) to SFs via EV, SFs were treated for 24 h with EV released from U937 cells upon stimulation with 5 µg/ml Poly(I:C) HMW Rhodamine or Poly(I:C) HMW Fluorescein. SFs were treated also with the Sup from the last washing of EV pellets or were stimulated with 5 µg/ml Rhodamine Poly(I:C) or 10 µg/ml Fluorescein Poly(I:C) for 24 h.
The presence of fluorescently-labeled Poly(I:C) in SFs was determined by flow cytometry and confocal microscopy. For flow cytometry, SFs were detached using the accutase, thoroughly washed with DPBS and immediately analyzed by FACSCalibur flow cytometer (BD Biosciences).
For confocal microscopy SFs, cultured in chamber slides (Lab-Tek; Nunc), were thoroughly washed with DPBS and fixed with 4% paraformaldehyde for 20 min RT. The slides were blocked with 1% bovine serum albumin/5% human serum for 40 min and incubated with FITC Mouse Anti-Human CD90 (BD Pharmingen, # 555595, 10 µg/ml, CD90 is a fibroblast surface marker) or FITC Mouse IgG1 κ isotype control antibodies (BD Pharmingen, # 555748, 10 µg/ml) for 1 h at RT. The nuclei were stained with DAPI (Sigma-Aldrich). Slides were covered with fluorescence mounting medium (Dako Cytomation). The images were taken by confocal laser scanning microscope Leica SP5 (Leica Microsystems) using the LAS AF software (Leica Microsystems).

CD 73 and 90 markers were selected using FACS for Human Bone Marrow Mesenchymal Cell Identification. PE mouse anti-human cd73 (BD Bioscience, cat.: 550257, 100tests) and FITC Mouse Anti-human CD90 (BD Bioscience, Cat: 555595) were used to identify cultured human MSC and cd31, cd73, cd135 and cd34 (BD Bioscience) were used for endothelia cells identification by FACS.