Cd45 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).

To analyze the surface markers of pADSCs by flow cytometry, cells were harvested with 100 μL of 0.25% trypsin-EDTA for 5 min at 37°C and washed twice in washing buffer (49.5 mL PBS and 0.5 mL FBS) by centrifugation at 400 × g. Cells (1 x 10⁵/100 μL) were incubated at 4°C for 30 min (covered with foil) with antibodies against phycoerythrin (PE)-conjugated CD29 (CD29-PE), CD31-PE, CD44-PE, CD45-PE, MHC I-PE and MHC II-PE (Sigma-Aldrich, St. Louis, MO, USA) for direct staining. The reaction was stopped by washing twice in 500 μL of washing buffer with 5-min 700-× g centrifugation, then fixed in fixing buffer (48.1 ml PBS + 0.5 mL FBS +1.3 mL of 37% formaldehyde) for flow cytometry analysis in the FACSCalibur system (BD Biosciences, New Jersey, USA) using CellQuest^™ software (Becton Dickinson, San Jose, CA, USA).

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).