Cd31 apc

Flow-cytometry analysis was performed on HUVECs, HSVECs and ECs-HU as follows. Cells were gathered, washed with PBS + EDTA (2 mM) + BSA (0.5%) and labeled in PBS + EDTA (2 mM) + BSA (0.5%) with CD31-APC, CD34-PE, CD144-PE and KDR-PE antibodies (Miltenyi, Bergisch Gladbach, Germany) for 10 min at 4 °C. The samples were analyzed by BD FACS Canto II flow cytometer (Becton–Dickinson, Heidelberg, Germany).

Cells were detached with trypsin, fixed with 4% PAF for 10min and then washed twice with PBS. Cells were re-suspended in PBS with 0.5% FBS. Cells were labeled with the following anti-human antibodies: CD105-APC, CD73-APC, CD90-APC, CD44-APC, CD34-APC, and CD31-APC (Miltenyi), CD45-APC (Becton Dickinson) for immunophenotyping assays; CD49a-APC and CD49d-APC (Miltenyi), CD106-APC and CD54-APC (Becton Dickinson) for adherence assays; Rabbit anti-p21, Mouse anti-p27, Mouse anti-Cyclin B1, Rabbit anti-Cyclin D1 (all from Cell Signaling), and Rabbit anti-p19 (Upstate) for cell cycle assays. Donkey anti-Mouse IgG DyLight650 and Donkey anti-Rabbit IgG DyLight650 (1:200 dilution for each, Thermo Scientific) were used as secondary antibodies when needed. Isotype antibodies served as respective controls. For intracellular labeling, cells were permeabilized with PBS/0.1% Triton X100 solution (BioRad). Cells were acquired on a FACS Scan flow cytometry analyzer (FACs Calibur, Becton Dickinson) and analyzed using CellQuestPro software (Becton Dickinson).

First-passage ASCs were labeled with CD13-APC-Vio700, CD31-APC, CD34-PE, CD45-VioGreen, CD73-PE-Vio770, CD105-VioBlue and CD146-VioBright515, purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Samples were acquired on a four-laser flow cytometry system (BD LSR II, Becton-Dickinson, Franklin Lake, NJ, USA). Fc receptor reagent was added to avoid unspecific labelling of cells via Fc receptors.

Freshly isolated cells were characterized for ADSVCs surface protein expression [17 (link)] by flow cytometry (MACSQuant analyzer, Miltenyi-Biotech) according to the manufacturer’s instructions. Cells were stained with the following antihuman-conjugated monoclonal antibodies: CD13-APC, CD14-PE, CD29-FITC, CD31-APC, CD34-PE, CD45-VioBlue, CD73-APC, CD90-FITC, CD105-VioBlue, CD144 (VE-Cadherin)-PE, CD146-Biotin, CD166-Biotin, HLA-ABC-FITC, HLA-DR-VioBlue, or relevant isotype-matched controls (Miltenyi-Biotech). Isotypes controls and automated compensation were settled to minimize false positive fluorescence and spectral overlap of fluorochromes respectively. Cell viability and apoptosis were assessed by the 7AAD/AnnexinV/PI assay. In fact, cell viability was first assessed manually using the Trypan blue (Sigma-Aldrich) exclusion assay, and the results were then validated by the 7AAD method by flow cytometry. The telomerase activity [18 (link)] was assessed by real-time qPCR (LightCycler 2.0, Roche, Basel, Switzerland) using the Quantitative Telomerase Detection Kit (Cat#MT3012, Allied Biotech, Taipei, Taiwan) according to the manufacturer’s instructions.

Cell sorting was performed using a MoFlo cell sorter equipped with SUMMIT software (Beckman Coulter, Buckinghamshire, UK). Following collagenase digestion of UC tissue, 2×10
⁶ cells were split into two tubes. One tube was stained with 5 μl of neat CD45-FITC (#F0861), CD235α-FITC (#F0870) (both from DAKO, Cambridge, UK), CD146-PE (BD Pharmingen) and CD31-APC (#130-092-652, Miltenyi Biotec), whereas the other was stained with 2.5 μl of neat isotype controls IgG1-FITC (#550617, BD Pharmingen), IgG1-PE (#MCA928PE, Serotec) and IgG1-APC (#130-098-846, Miltenyi Biotec). After incubation with relevant antibodies and washes, 2 μg/ml 7-aminoactinomycin D (7-AAD) (#A1310, Invitrogen) was added to exclude dead cells before sorting into four fractions: haemopoietic cell fraction (HC), CD45
⁺CD235α
⁺CD31
^-; EC fraction, CD45
^-CD235α
^-CD31
⁺; candidate MSC fraction, CD45
^-CD235α
^-CD31
^-CD146
⁺ and non-MSC fraction, CD45
^-CD235α
^-CD31
^-CD146
^-. The latter two subsets were processed for telomere length analysis.