Resveratrol in IL-2 Therapy against Melanoma Figure 3. Resveratrol protects endothelial integrity. The ultrastructural studies showed resveratrol protects endothelial cells. Panels 1 and 2 show PBS- or resveratrol-treated mice, respectively. The endothelial cell (En) lining the blood vessel is firmly anchored to the basal lamina (BL) with good integrity, thereby showing normal morphology. The cell has an intact nucleus (Nu) and is closely opposed to the intact basal lamina. Three RBCs lie within the blood vessel lumen. Panel 3 shows IL-2-treated mice with significant damage to the endothelial cells. Cell membrane is folding (F) and discontinuous (D). Vacuolation (V) is found under the folded cell membrane and within the cytoplasm. In addition, some endothelial cells have lost the normal morphology, with only extended cell membrane remnants remaining. Cellular debris from former endothelial cells can be found in the blood vessel lumen. An RBC fills the lumen. Panel 4 shows IL-2+resveratrol-treated mice with normal endothelial cell morphology. The cell membrane and cytoplasmic contents are well defined and closely adhere to the basal lamina. The intact nucleus is closely opposed to the intact basal lamina Three RBC fill the lumen. Original magnification, 620,000. doi:10.1371/journal.pone.0035650.g003 purified using NycoPrep and referred to as LAK cells. Cytotoxicity of LAK cells was tested against B16F10 tumor or TME endothelial cells using 4 h 51Cr-release assay. Briefly, 16106 target cells were labeled with 100 mCi of 51NaCrO4 (MP Biomedicals) at 37uC for 1 h, washed three times in RPMI 1640 culture medium, and adjusted to 16105 cells/ml. In brief, 16104 target cells were plated into 96-well U-bottom plates with varying numbers of LAK cells depending on the respective E:T cell ratios. In these experiments, the LAK cells were defined as the effector cells (E) that mediate lysis of the 51NaCrO4-labeled endothelial cells or tumor cells, which were defined as the target cells (T). In some groups, target cells were incubated with resveratrol (25 mM) for 2 h at 37uC before they were added into the plates. For analysis of MDSC suppression, LAK cells were incubated with MDSC for 2 h before adding to the plates. Spontaneous release of 51NaCrO4 was determined by culturing the target cells alone, and the maximum release of 51NaCrO4 was determined by incubating the target cells with 1% SDS. The supernatants were harvested after 4 h culture, and the radioactivity was measured using a Microbeta counter (PerkinElmer). Percentage of killing efficiency of LAK cells was calculated as percent cytotoxicity = [(sample cpm 2 spontaneous release cpm)/(maximum release cpm 2 spontaneous release cpm)] 6100. The data were represented as mean 6SEM. Each group had four wells. Isolation of T cells and infiltrating inflammatory cells To isolate T cells from spleen, splenocytes were stained with anti-CD3 Abs, followed by magnetic beads isolation. For isolation of infiltrating mononuclear cells (MNCs) from lung or liver, mice were perfused. Lung or liver was minced and passed through 100 mm filter. Single-cell suspensions were subject to Percoll gradient (70%/30%) centrifugation. TUNEL assay of cell apoptosis Cell apoptosis was evaluated with conventional TUNEL assay using DeadEnd Colorimetric TUNEL System from Promega by following the manufacturer’s instructions. Numbers of TUNEL- PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35650