Facs sort

The lungs and spleens were polished into pieces. Then the lymphocytes collect were extracted with the Lymphocyte Separation Medium (Shenzhen, China) according to the manufacturer's protocol. Percentages of NK cells were evaluated and separated with flow cytometry using monoclonal antibodies (MoAbs) anti-CD3⁻ FITC/NK1.1⁺ PE (BD Phamingen, San Di ego, CA). During analysis, the CD3⁻/NK1.1⁺ Population was determined. To determine the surface expression of the NK cell ligands on NK cells, the cells were then stained with the goat anti-mouse NK1.1-FITC, CD3- Alexa Fluor 647, NKG2A-FITC, NKG2D-PE, Ly49I-PE (BD Phamingen, San Di ego, CA) for 30 min at 37°C in the dark. The analysis was performed on the FACS Sort (Becton Dickinson, Mountain View, CA) using Cell Quest software (Becton Dickinson) and the cell surface expression was quantified by the value of the mean fluorescence intensities obtained with the specific mAbs.

To determine the surface expressions of NKG2D ligands on cancer cells, the cells were incubated with mouse anti-MICA, anti-MICB, anti-ULBP1-3 (R&D systems, Minneapolis, MN, USA), anti-HLA-ABC (Clone W6/32, Serotec, Oxford, UK) or the corresponding isotype controls at 10 μg/ml and then incubated with goat anti-mouse-PE conjugated (BD Pharmingen Inc., San Diego, CA., USA). The analysis was performed on a FACS Sort® (Becton Dickinson, Mountain View, CA., USA) using Cell Quest software (Becton Dickinson), and cell surface expressions were quantified using mean fluorescence intensities (MFIs). Relative expression ratios were calculated by dividing treated sample MFI by untreated sample MFI without subtracting the MFI of the appropriate isotype control.

Commercially available ELISA kits were used to quantify plasma levels of sCD14, IL-6, and TNF-α (R&D Systems Europe, Abingdon, United Kingdom). The assays were performed in duplicate according to manufacturer’s instructions. In a subset of patients with an available frozen cell sample stored in viable conditions, surface phenotypes were evaluated on thawed PBMCs by flow cytometry (FACS Sort Becton-Dickinson, San Josè, California, USA) using directly labeled antibodies (fluorescein isothiocyanate [FITC], phycoerytrin [PE], and Peridinin-chlorophyll-protein complex cyanin 5.5 [PerCP Cy5.5]). We determined CD8+ T-lymphocyte activation by measuring the expression of HLA-DR and CD38. The following combination was used: CD8/CD38/HLA-DR (CD38-PE, HLA-DR-FITC CD8-PerCP Cy5.5, Becton Dickinson, San Josè, CA, USA). All biomarkers were measured in a central laboratory and all technicians were blinded to patients’ disease progression status.
Plasma concentrations of LPS were determined with a commercial LAL kit (Kinetic-QCL; Bio Whittaker, Walkersville, MD, USA) for the quantitative determination of LPS using a known endotoxin standard as a reference (E. coli O55:B5 endotoxin). Plasma samples were diluted with endotoxin-free water and then heated to 90°C for 15 min to inactivate plasma proteins. The assay was carried out according to the protocol recommended by the manufacturer.

Fresh NK cells were obtained from normal healthy donors with informed consent in accordance with the Declaration of Helsinki. Target cells (1X10⁵ cells/ml) were stained using the Vybrant® carboxyfluorescein diacetate, succinimidyl ester (CFSE) Cell Tracer Kit (Invitrogen, Eugene, OR, USA) and incubated with NK-92 cells or freshly isolated NK cells at selected effector/target ratios for 2 hours in 5 ml round-bottomed tubes. These co-cultured target cells and NK cells were then stained with 1 μg/ml propidium iodide (Sigma-Aldrich). The assay was performed on FACS Sort® (Becton Dickinson) by acquiring 3,000 target cells. The percent if specific release was calculated by the number of PI⁺&CFSE⁺ cells/3,000 X 100 (%). All experiments were performed in triplicate.

The effect of DPEITC (72h) on cell cycle progression was determined by PI staining followed by analysis using a Becton Dickinson FACS sort (BD Biosciences, San Jose, CA, USA) and Mod Fit program (Verity Software House, Topsham, ME, USA), as described previously [20 (link),21 (link)]. Briefly, cells were harvested after treatment by centrifugation at 190× g for 3 min at 4 °C, washed once with PBS, and stored as a suspension in 1 mL of 70% ethanol at –20 °C overnight. Cells were then harvested by centrifugation at 420× g for 10 min and resuspended in 1 mL of freshly prepared PI staining solution (PBS with 0.1% Triton X-100, 0.05 μg/mL propidium iodide, 0.1 mg/mL RNase (Sigma)). The cell suspension was incubated at RT for 30 min in the dark, followed by incubation for 30 min at 4 °C before analysis. Similarly, cells were treated with DMSO, DPEITC, ATZ, DPEITC, and ATZ, CPT, or DPEITC and CPT for indicated periods of time and cell cycle analysis was performed, as described above.

A total of 1×10⁶ cells/well were treated with resveratrol for 24 h and double-staining with Annexin V-FITC (1 μl) and PI (1 μg) was performed. The cells were then washed with phosphate-buffered saline (PBS; Beyotime, Haikou, China) and analyzed by flow cytometry (FACS sort; BD Biosciences).