Anti cd105 pe

According to methods previously reported,²⁵, ²⁶ ADSCs were isolated from human adipose tissue obtained from patients who were undergoing liposuction at the Department of Plastic Surgery, the First Hospital of China Medical University. Adherent cells were cultured in a growth medium [DME F12 (HyClone, USA), 10% FBS, 1% Penicillin‐Streptomycin Solution (Gibco, USA)] at 37°C/5% CO2 and saturated humidity. ADSCs were passaged after reaching 90% confluence.
Multi‐lineage potential assay and flow cytometry analysis were performed to identify characteristics of ADSCs, as we have done in the past.⁴ The antibodies including anti‐CD34‐FITC, anti‐CD45‐PE, anti‐CD44‐FITC, anti‐CD73‐PE and anti‐CD105‐PE were purchased from BD biosciences (USA).

Flow cytometry was performed using a FACSCalibur Cytometer (BD Biosciences); a phenotyping kit (MSC phenotyping kit, Miltenyi) was used to characterize the tMSCs. The following antibodies were used: anti-CD34PerCP, anti-CD45PerCP, anti-CD20PerCP, anti-CD14PerCP, anti-CD73APC, anti-CD9FITC, and anti-CD105PE (BD Pharmingen). Matched isotype controls were applied to determine background fluorescence levels.

The immunophenotypes of the BM-DFATs, SC-DFATs, BM-MSCs, ASCs at passage 2 were identified using flow cytometry as previously described [13 (link)]. The cells grown to 60% confluence were suspended at a density of 5 × 10⁵ cells per tube and incubated with various anti-human antibodies conjugated with phycoerythrin (PE) or allophycocyanin (APC). The following antibodies were used: anti-CD73-PE, anti-CD90-APC, anti-CD105-PE, anti-CD31-PE, anti-CD45-APC, anti-HLA-DR-PE, anti-CD106-PE, anti-CD54-APC, and anti-CD36-PE (all from BD Biosciences, San Jose, CA). Mouse IgG1-PE, mouse IgG1-APC, mouse IgG2a-PE, mouse IgG2b-APC, and mouse IgM-PE (all from BD Biosciences) were used as negative controls. The fluorescence intensity of the cells was evaluated by a FACSAria flow cytometer (Becton Dickinson, Bedford, NJ), and data were analyzed using FlowJo software (version 10.6.1, FlowJo, Ashland, OR). Positive cells were counted and compared with the signal of corresponding immunoglobulin isotypes. A minimum of 1 × 10⁴ events were recorded for each sample, and analysis was performed at least three separate times for each condition tested.

Flow cytometry was performed on matched, culture-expanded SF-MSCs and Sm-MSCs (n = 3 donors) using a standard panel of markers to define cultured MSCs^{10 (link)} through use of an LSRII 4-laser flow cytometer (BD Biosciences). The following antibodies and appropriate isotype controls were used: anti-CD34-allophycocyanin-cyanine (APC), anti-CD19-phycoerythrin (PE), anti-CD45-phycoerythrin-cyaine (PE-Cy7), anti-CD14-fluorescein isothiocyanate (FITC), anti-CD73-PE, anti-CD90-PE-Cy7, and anti-CD105-PE (all from BD Biosciences). Dead cells were discriminated by use of 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). At least 10,000 live cell events were collected for each antibody combination.

Passage 3 ADSCs were digested by trypsin-EDTA and washed with PBS, then incubated with fluorescein isothiocyanate- (FITC-) conjugated and phycoerythrin- (PE-) conjugated antibodies, including anti-CD34-FITC, anti-CD44-FITC, anti-CD90-FITC, anti-CD45-PE, anti-CD73-PE, anti-CD105-PE, PE-labeled mouse IgG1 Kappa, and FITC-labeled mouse IgG1 Kappa (BD Pharmingen, USA) at 4°C for 40 min. We used flow cytometry (LSRFortessa, BD Biosciences, USA) to record and analyze the data. At least 1 × 10⁴ cells were analyzed per test.
Briefly, 12-well plates were pretreated with gelatin (Cyagen, USA) to enhance adherence. Then, to confirm the multilineage differentiation ability of ADSCs, passage 3 cells were seeded at a density of 1 × 10⁴ cells/well and cultured until 60–70% confluence was achieved. The medium of some of the wells was then changed to mesenchymal stem cell osteogenic differentiation medium (Cyagen, USA) according to the manufacturer's instructions. The medium was changed within 72 h. For adipogenic differentiation, we used mesenchymal stem cell adipogenic differentiation medium (Cyagen, USA) according to the manufacturer's instructions. Two to four weeks later, the cells were stained with alizarin red S (Solarbio, China) and oil red O (Cyagen, USA) separately to identify osteogenic and adipogenic differentiation, respectively.

Anti-active-β-catenin and β-catenin antibodies were purchased from Millipore (Temecula, CA, USA). Anti-CD105-PE, anti-CD146-PE, anti-CD90-PE, anti-CD34-PE, and anti-CD45-PE antibodies were purchased from BD Bioscience (San Jose, CA, USA). Unconjugated anti-GSK3β and anti-phospho-GSK3β antibodies were purchased from Cell Signaling Inc. (San Francisco, CA, USA). Anti-β-actin antibody was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Unconjugated anti-CBS, CSE, DSPP (DSP)  and TRPV1 were purchased from Abcam Inc. (Cambridge, MA, USA).

The cells were trypsinized and washed twice in PBS (PanEco, Moscow, Russia) supplemented with 1% FBS (Gibco, Waltham, MA USA) by centrifugation for 5 min at 300× g. The cells were incubated with anti-CD14-FITC, anti-CD34-APC, anti-CD45-PE, anti-CD29-APC, anti-CD44-FITC, anti-CD73-PE, anti-CD105-PE, anti-CD95-APC (BD Biosciences, Franklin Lakes, NJ, USA) at concentrations recommended by the manufacturer, for 1 h at 4 °C in the dark. At the end of the incubation, the cells were washed twice in PBS by centrifugation. Fluorescence intensity was analyzed using BD FACSAria III flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). At least 10⁴ events were recorded in each sample. The results were processed using the FlowJo_V10 (FlowJo™, Ashland, OR, USA). The relative fluorescence intensity (RFI) of marker expression was calculated as the ratio of the specific fluorescence of cell staining with fluorescently labeled antibodies and the autofluorescence of control unstained cells.