Biosciences facscalibur flow cytometer

Cells at passage 3 (p3) were detached by using trypsin-EDTA 0.2% (Gibco, Carlsbad, CA, USA), fixed with methanol for 10 min at −20°C, washed with 1% BSA in PBS, and then incubated with permeabilization buffer: 0.1% Triton X-100 and 1% BSA in PBS for 10 min at 4°C. Cells were incubated with antibodies raised against CD31, CD34 CD45, CD73, CD90, CD105, CD146, and HLA-DR (Santa Cruz Biotechnology, Dallas, TX, USA) on ice for 30 min. Cells were pelleted, washed, and fixed in 1% paraformaldehyde. Fluorescence-activated cell sorting (FACS) analysis was performed on BD Biosciences FacsCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) using FlowJo software for analysis.

The analysis of protein expression were performed as previously published [15 (link), 16 (link)]. In brief, the Hepa1c1c7 cells (1 × 10⁵ cells/well, the cell density reached 80 to 90% confluence), were treated with PHO-S (0.3–2.0 mM), DODAC/PHO-S 1:1 (0.3–2.0 mM), and empty DODAC (0.3–2.0 mM), for 12 h, were washed with PBS and resuspended in FACS buffer with 2.5% paraformaldehyde for 1 h. After washing, cells were again resuspended in a primary antibody specific for the proteins anti-CD44, anti-CD90, anti-p53, anti-p21, anti-p27, anti-Bax, anti-Bcl-2, anti-caspase-3, anti-caspase-8 (Abcam, Cambridge, MA, United States); anti- cytochrome c, anti-DR4 (Santa (Cruz Biotechnology Inc., Santa Cruz, EUA) and anti-cyclin D1 (Cell Signaling Technology, Danvers, MA), at a concentration of 1 μg/ml at 4 °C, for 1 min. The corresponding isotope antibody was used as a negative control and as a secondary antibody was used Goat anti Mouse IgG (H/L): FITC (AbD Serotec, Raleigh, NC, United States). The cells were pelleted, washed twice with PBS, then, fluorescence-activated cell sorting (FACS) analysis was performed on BD Biosciences FACs Calibur flow cytometer (Becton Dickinson, San Jose, CA, United States) using Cell Quest and Win MDI 2.9 softwares.

The cellular pellet was resuspended in phosphate-buffered solution (PBS) at a concentration 5 × 10⁵ cells/mL and incubated for 1 h at 4°C with 1 μl of specific antibodies: cyclin D1 conjugated with fluorescein isothiocyanate (Abcam, Cambridge, MA, United States) and specific antibody caspase-3 active with phycoerythrin (PE) (Abcam, Cambridge, MA, United States), in absence or presence of an Ac-Asp-Glu-Val-Asp-OH-specific inhibitor (Abcam, Cambridge, MA, United States). After this period the cells were centrifugate at 428 × g for 10 min, washed twice with PBS-cold and fixed with 1% paraformaldehyde. Flow cytometer settings were established using unstaining cells. Cells were gated by forward scatter to eliminated debris. To eliminate the possible autofluorescence of ADSCs, the contribution of unstained cells in the measurement channel was removed. A minimum of 10,000 events was counted for each analysis. Cells were evaluated for cell surface proteins expression using BD Biosciences FacsCalibur flow cytometer (Becton Dickinson, San Jose, CA, United States) using Cell Quest, and Win MDI 2.9 softwares was used to create the histograms.

Peripheral whole blood samples (150 µL) from 21 patients were immunophenotyped by flow cytometry, with previously titrated monoclonal antibodies (Becton-Dickinson, San Jose, CA, USA, EUA; Figures S1–S3 in Supplementary Material). Cells were analyzed with BD Biosciences FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA, EUA). Gating strategies are shown (Table S1 in Supplementary Material). Thirty-thousand events per sample were acquired for each subset and 100,000 events for immunoregulatory T cells. Results were expressed as absolute cell numbers (cells per microliter).