Fitc mouse anti human cd105

Collected MSCs were incubated for 30 min at room temperature with the following specific antibodies: PE mouse anti-human CD29, FITC rat anti-human CD44, FITC mouse anti-human CD105, FITC rat anti-human CD45, APC mouse anti-human CD34, and PE mouse anti-human HLA-DR (all from BD Pharmingen). As a control, the cells were stained with the appropriate isotype antibodies. At the end of co-culture, the CD4⁺ T cell apoptosis was analyzed by using an Annexin V-PE Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer’s instructions. To detect Treg cells, a Human Regulatory T Cell Staining Kit (eBioscience) containing an anti-CD4-FITC/CD25-APC cocktail and anti-Foxp3-PE was used according to the manufacturer’s instruction. In addition, we used a Human Th1/Th2/Th17 Phenotyping kit (BD Pharmingen) to analyze the T helper cell subsets. All samples were analyzed using a BD Biosciences Influx cell sorter.

H9‐derived MSCs and human bone marrow‐derived MSCs (Lonza, Walkersville, MD,
http://www.lonza.com) were grown to confluence, harvested by using 0.25% trypsin/EDTA, and resuspended in buffer containing phosphate‐buffered saline (PBS), 2% HEPES buffer, 2% FBS, and 0.1% bovine serum albumin (BSA) as previously described 43. Cells (1 × 10⁶) were incubated with phycoerythrin (PE) mouse anti‐human CD90, PE mouse anti‐human CD73, fluorescein isothiocyanate (FITC) mouse anti‐human CD44, FITC mouse anti‐human CD45, FITC mouse anti‐human HLA‐ABC, PE mouse anti‐human CD29, PE mouse anti‐human CD166, PE mouse anti‐human HLA‐DR, FITC mouse anti‐human CD105, or FITC mouse anti‐human CD31 (BD Biosciences, San Jose, CA,
http://www.bdbiosciences.com). Nonspecific fluorescence was determined by using isotype‐matched monoclonal antibodies. A total of 10,000 events were collected on a BD fluorescence‐activated cell sorting Calibur Flow Cytometer instrument by using CellQuest software (BD Biosciences). Analyses of results and corresponding graphs were generated by using FlowJo software (Tree Star, Ashland, OR,
http://www.flowjo.com) 434445.

Dental pulp tissues were isolated from extracted deciduous incisors (6–8-year-old donors). Parents of these donors signed written informed consent forms. Pulp tissues were isolated, washed, digested in 3 mg/ml collagenase type I (Sigma-Aldrich, United States) for 2 h and incubated with culture medium at 37°C in 5% CO₂. At 80% confluence, the cells were trypsinized and subcultured. The specific cell surface molecules were identified using flow cytometry. Briefly, cells at passage three were trypsinized and incubated with FITC mouse anti-human CD105 (Cat.561443), PE mouse anti-human CD34 (Cat.555822), PE mouse anti-human CD90 (Cat.555596), PE mouse anti-human HLA-DR (Cat. 555812) and BV510 mouse anti-human CD45(Cat.563204) (all from BD Biosciences, United States) on ice for 30 min. Then the cells were washed, resuspended and detected with a flow cytometry system (BD LSRFortessa, BD Biosciences, Franklin Lakes, NJ, United States).