Resveratrol in IL-2 Therapy against Melanoma Figure 2. Decreased VLS and increased tumor regression in resveratrol-treated melanoma-bearing mice. C57BL/6 mice were injected i.v. with B16F10 melanoma cells (56105 cells/mouse) and adminsitered resveratrol as described in Fig. 1. On day 9, mice were treated with IL-2 as in Fig. 1. The mice were sacrificed for evaluation of VLS on day 12. The numbers of black metastatic nodules on the lung surfaces were counted under a microdissecting microscope. The area of each nodule was measured with software Image Pro. The values are shown as mean 6SEM. A. VLS in lung. B. VLS in liver. C. Enumeration of lung metastatic nodules. D. Area measurement of lung metastatic nodules. E. Histopathology of lung metastasis on the surface of one lobe. doi:10.1371/journal.pone.0035650.g002 MDSC sorting and adoptive transfer MDSC were sorted by two steps. First, MDSC cells were enriched from splenocytes of VLS mice by depletion of other cells (CD4+, CD8+, CD90.2+, B220+, NK1.1+, F4/80+, CD11C+, CD117+) with magnetic beads, and then, CD11b+Gr-1+ double positive cells were sorted by a flow cytometer. Briefly, splenocytes at a density of 16108 cells/ml in the staining buffer were pre- incubated for 10 min with purified anti-CD16/32 antibody to block non-specific binding of antibodies to Fcc receptors, and then incubated with biotin-conjugated anti-mouse CD4, CD8, CD90.2, B220, NK1.1, F4/80, CD11c, and CD117 mAbs for 15 min at room temperature. Cells were washed once with and re-suspended in the staining buffer at the same cell density. Cells were then incubated with EasySep biotin cocktail (Stemcell) at room temperature for 15 min followed by incubation with EasySep magnetic particles (Stemcell) at room temperature for 10 min. Non-magnetic binding cells were collected, and further incubated with FITC-conjugated anti-mouse CD11b and PE-conjugated anti-mouse Gr-1 mAbs for 30 min at 4uC. Cells were washed twice with and re-suspended in the staining buffer. CD11b and Gr-1 double positive cells were then sorted by a FACSAria sorter. The purity was .98%. For adoptive transfer, 16106 cells were transferred per recipient mouse intravenously. Generation of LAK cells and 51Cr release assay LAK cells were prepared as previously described [12,26,27,28]. For generation of LAK cells in vitro, splenocytes from na¨ıve mice were cultured for 48 h with 1000 units/ml IL-2 in RPMI containing 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acid, 100 units/ml penicillin, 100 mg streptomycin, and 50 mM b-mercaptoethanol. Viable cells were purified by density gradient centrifugation using NycoPrep (Cedarlane, Burlington, NC). Such cells will be referred to as LAK cells. For generation of LAK cells in vivo, mice were treated with IL-2 as described above. On day 4, splenocytes were prepared and viable cells were PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35650