Tein content of your cell lysates was measured plus the volume of intracellular doxorubicin was detected working with a PerkinElmer LS-5 spectrofluorimeter (PerkinElmer). Excitation and emission wavelengths were 475 and 553 nm, respectively. A blank was prepared within the absence of cells in each set of experiments and its fluorescence was subtracted in the one particular measured in every sample. The results had been expressed as nmol doxorubicin/mg cell proteins based on a previously prepared calibration curve.Dendritic cell (DC) generation and in vitro phagocytosis assayCells have been washed twice with PBS, rinsed with 1 ml of 0.25 (w/v) PBS-BSA and centrifuged at 10,000 g for 5 min. The pelleted cells had been incubated for 45 min at 4 with the anti-Pgp, anti-MRP1, anti-BCRP or anticalreticulin antibodies, then washed and incubated using a secondary FITC-conjugated antibody for 30 min at 4The generation of DCs and their phagocytosis of colon cancer cells had been performed as described earlier [26].Resorufin Cancer DCs had been generated from peripheral blood samples obtained from wholesome donors kindly provided by the regional Blood Bank (Fondazione Strumia, Torino, Italy). Cells have been harvested on day 6 and confirmed as immature DCs by morphology and immunophenotype (information not shown). HT29 and HT29-dx cells were green-stained with PKH2FITC (Sigma), washed twice and incubated with DCs at a ratio of 1:1 for 18 h at 37 . Co-cultures have been then stained for 20 min at 4 with APC-conjugated HLA-DR antibody (Miltenyi Biotec, Tetrow, Germany) to mark DCs. Twocolor flow cytometry was performed with FACS-Calibur method and CellQuest software program. At least ten,000 events had been accumulated particularly backgating on DC morphology (area 1: FSC versus SSC). Tumor cell phagocytosis was assessed because the percentage of double-stained (FITC plus APC) cells. Tumor cells do not express important amounts of HLA-DR, and they have been excluded from area 1 by their morphology. In every single set of experiments, a phagocytosis assay was performed by co-incubating DCs and tumor cells at four , instead of 37 , along with the percentage of double-stained cells obtained just after the incubation at four was subtracted from values observed at 37 . The phagocytosis price was expressed as “phagocytic index”, calculated as reported in [42].Gelsomino et al. Molecular Cancer 2013, 12:137 http://www.molecular-cancer/content/12/1/Page 18 ofStatistical analysisData are reported as mean SD of a minimum of 3 independent experiments. Final results have been checked for standard distribution and analyzed by a one-way evaluation of variance (ANOVA) followed by Dunnet’s t test.Maltohexaose Endogenous Metabolite p 0.PMID:25147652 05 was thought of significant.Extra filesAdditional file 1: Effects of 3PUFAs on apoptosis and necrotic/ immunogenic death of colon cancer cells. HT29 and HT29-dx cells were incubated for 24 h in the absence (CTRL) or presence of 50 M arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA). Cycloheximide (four M for 24 h, CHX) was selected as constructive handle of cytotoxicity in each chemosensitive and chemoresistant cells; doxorubicin (five M for 24 h, DOX) was chosen as optimistic control of cytotoxicity in chemosensitive cells. A. Annexin V staining. The percentage of cells optimistic for surface annexin V, taken as index of apoptosis, was measured in duplicate by flow cytometry. Data are presented as signifies SD (n = 3). Versus respective CTRL: * p 0.001; DOX in HT29-dx versus DOX in HT29: p 0.001. B. Western blot evaluation of extracellular HMGB1, taken as index of necrotic/immunoge.