Cd105 pe

In order to analyze the cell surface antigen
expression, we used trypsin-EDTA to harvest 5×105
fresh passage-3 cells. Cells were centrifuged at 100
g for 1 minute, resuspended in stain buffer (PBS,
2% FBS) and incubated on ice for 10 minutes.
Trypsin was neutralized by centrifugation; isolated
cells were washed twice with PBS and resuspended
in stain buffer. Cells were incubated in the dark
for 30 minutes. After incubation, the cells were
labeled with the following anti-human monoclonal
antibodies (MAbs) conjugated to fluorochromes:
anti-CD90-FITC, CD73-PE, CD11b-FITC, CD34-
FITC, CD44-FITC, CD45-PE, and CD105-PE
(Sigma-Aldrich, USA). The frequencies of all
immunolabeled cells were analyzed by a FACS
Canto II flow cytometer (BD Biosciences, USA),
in which approximately 500,000 events were
assessed. Data were analyzed by FlowJo software
(version 10.0).

The 3–5 generation adhered cells were digested with 2.5 g/l trypsin, and when the cells were nearly round under an inverted microscope, DMEM/F12 medium containing 10% fetal bovine serum (FBS) was added to terminate the digestion, followed by 5 min centrifugation at 840 × g for 5 min and twice washing with phosphate-buffered saline (PBS). PBS-balanced solution (100 μl) was added to prepare the cell suspension. Mouse-anti-human monoclonal and negative-control antibodies were added (all purchased from Sigma-Aldrich, St. Louis, MO, USA): i) Cluster of differentiation 105-phycoerythrin (CD105-PE) and immunoglobulin G (IgG)1k-PE. ii) CD29-fluorescein isothiocyanate (FITC) and IgG2a-FITC. iii) CD44-FITC and IgG1-FITC. iv) CD14-FITC and IgG2ak-FITC. v) CD34-PE and IgG1k-PE. vi) CD45-PE-cyanine 5 (PECY5) and IgG1-PECY5. The cells were incubated for 30 min at room temperature, washed twice with PBS following centrifugation, followed by the addition of FITC cross-linked rat-anti-mouse second antibody (1:400). Subsequent to culture for 45 min, the cells were resuspensed with 3 ml PBS, washed with PBS at 840 × g for 5 min and resuspended in 300 μl PBS. The cell suspension was filtered and analyzed using flow cytometry. All the procedures were repeated three times.

We used the following materials in this clinical
trial study: collagenase A type I (Sigma, USA), fetal
bovine serum (FBS, Gibco, USA), MEM Alpha
1x (Gibco, USA), L-glutamine (Gibco, USA),
antibiotic-antimycotic solution (Gibco, USA),
trypsin-EDTA (Gibco, USA), CD90-fluorescein
isothiocyanate (FITC), CD73-phycoerythrin (PE),
CD105-PE, CD34-FITC, CD45-PE, CD11b-FITC,
CD44-FITC (Sigma-Aldrich, USA), colcemid
solution (Invitrogen, USA), and dimethylsulfoxide
(DMSO, Gibco, USA). One-way ANOVA test was
used in this study which was not significant (P>0.05).