Facscalibur flow cytometry system

At 48 hours after transfection, the CD4⁺ T cell suspension (100 μl; 1 × 10⁵ cells) was incubated with fluorescein isothiocyanate (FITC)-conjugated anti-human CD11a (Becton Dickinson, San Jose, CA, USA) for 20 min at room temperature, washed in 2 ml of Stain Buffer (pH 7.4) containing 0.2% (w/v) bovine serum albumin (BSA) (Becton Dickinson, San Jose, CA, USA), and centrifuged at 400 x g for 5 min. The pelleted cells were subsequently resuspended in 0.5 ml of PBS/BSA. The FACSCalibur flow cytometry system (Becton Dickinson, Franklin Lakes, NJ, USA) was used to acquire fluorescence signal, and the results were subsequently analyzed using CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). Relative CD11a protein expression is shown as the MFI and the percentage of cells expressing CD11a.

The generation ROS was measured using a dihydroethidium (DHE) probe (Beyotime). Briefly, cardiomyocytes were separated and incubated with the DHE (50 μM) probe for 30 min at 37°C. Levels of ROS were analyzed using a FACSCalibur flow cytometry system (Becton Dickinson & Co., D) with an argon‐ion laser tuned to 485 nm.

Isolated hTGSCs, hDPSCs, and hPDLSCs (passage 3) were characterized for their mesenchymal cell surface profiles, as described previously.¹⁶ (link)^–18 (link) The hTGSCs and hDPSCs were trypsinized and incubated with the following conjugated antibodies: CD29, CD34, CD45, CD73, CD90, CD105, CD133, and CD166 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The hPDLSCs were then incubated with primary antibodies raised against STRO-1, CD146, CD90, CD44, CD19, or CD14. The cells were washed with phosphate-buffered saline (PBS) to remove the excess primary antibodies. The cells were analyzed by flow cytometry using a FACSCalibur flow cytometry system (Becton Dickinson, San Jose, CA, USA).

Apoptotic cell death was detected by Annexin-V–Allophycocyanin (APC)/Propidium Iodide (PI) double staining (Immunostep, Salamanca, SP, EU), according to the manufacturer’s instructions. This staining allows quantification of early (annexin-V⁺/PI⁻) and late (annexin-V⁺/PI⁺) apoptotic cells, as well as necrotic cells (annexin-V⁻/PI⁺). In order to detect cell death, cells were recovered after 24 h, double stained with Annexin-V and propidium iodide and analyzed on a FACSCalibur flow cytometry system (Becton-Dickinson, San Jose, CA, USA) using BD CellQuest software version 3.3 (Becton-Dickinson, San Jose, CA, USA). Data were analyzed using FlowJo 7.3.5 software for Windows (Tree Star, Inc. Ashland, OR, USA).

Cell cycle analysis was performed using flow cytometry following the harvesting and washing of the cells with cold PBS. Briefly, the cells were fixed in 75% ethanol and stored at −20°C for subsequent analysis. The fixed cells were subjected to centrifugation at 200 × g at 4°C for 5 min and washed twice with cold PBS. Ribonuclease A (final concentration, 20 μg/ml) and propidium iodide staining solution (final concentration, 50 μg/ml; Promega Corp.) were added to the cells, which were subsequently incubated for 30 min at 37°C in the dark. Cell analysis (≥100,000 cells) was performed using a FACSCalibur flow cytometry system (Becton Dickinson) equipped with CellQuest™ 3.3 software. ModFit™ LT 3.1 trial cell cycle analysis software (Verity Software House, Topsham, MA, USA) was used to determine the proportion of the cells in each of the different phases of the cell cycle.

Detection of Strol-1⁺, the cell surface marker, was performed by trained technicians blinded to patient identity using Becton Dickinson FACS Calibur Flow Cytometry System (Becton Dickinson, Beckman Coulter, Brea, CA, USA) equipped with Cell Quest software (Beckman Coulter). BMSC cell suspension was incubated with primary antibody for 1 h at 4 °C. Unbound antibodies were removed by washing with PBS. The secondary monoclonal antibodies conjugated with allophycocyanin (APC) were used to detect Stro-1⁺ (BD Pharmingen, San Diego, CA, USA) (1:100 dilution). After incubation, cells were washed and resuspended in 500 L of wash buffer and measured by FACS. The signals corresponding to debris and cell aggregates were first gated out by using the forward light scatter (FSC) and side light scatters (SSC) display. Furthermore, absolute counts of Stro-1⁺ positive cells in BMSC were determined using BD TruCOUNT Tubes (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. During analysis, the absolute number of Stro-1+ positive cells in cultured BMSC was manually calculated using the following equation: (events of Stro-1⁺ positive cells/events of beads) * (number of beads per test/test volume).

The DPMSCs, UCMSCs, and ADMSCs were checked for their surface marker profile by FACSCalibur flow cytometry system (Becton Dickinson, CA, USA) as already described [8 (link), 9 (link), 23 (link)]. Briefly, the MSCs were detached from the surface of the flask with enzyme digestion for 3 minutes at room temperature, collected, and centrifuged at 300g for 5 minutes. The pellets were resuspended in stain buffer and the cells were counted by hemocytometer. Then, 2.5 × 10⁵ cells were incubated for 45 min, in the dark at 4°C, with the following antibodies: fluorescein isothiocyanate- (FITC-) labeled mouse antihuman CD90 (StemCell Technologies, Milan, Italy), CD105, CD14, and CD19 (Diaclone, France); R-phycoerythrin- (PE-) labeled mouse antihuman CD34, CD44, CD45 (Diaclone, France), and CD73 (Becton Dickinson, CA, USA); and anti-HLA-DR (Diaclone, France). The control for FITC- or PE-coupled antibodies was isotypic mouse IgG1. The data were evaluated using CellQuest software (Becton Dickinson, CA, USA).