Cd34 fitc

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).

To analyze the expression of surface markers of the stem cells, flow cytometry was performed. At least 200,000 cells were incubated with fluorescencelabeled monoclonal antibodies against CD29-PE, CD34-FITC, CD44-PE and CD45-FITC (Sigma Aldrich, USA) Following a 10 minutes wash in PBS, the labeled cells were analyzed using a FACS Calibur flow cytometry apparatus (2 (link),20 (link)).

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).