Figure 5. Assay analyzing cytotoxic effects of liver lymphocytes obtained from fasted mice on tumor cells.

(A) The cytotoxic activity of freshly isolated liver lymphocytes from fed mice (solid lines) and 3-day-fasted mice (dashed lines) against TRAIL-resistant YAC-1 and (B) TRAIL-sensitive Hepa1-6 cells was analyzed using the ⁵¹Cr-release assay. The effector to target (E/T) ratios were 40∶1, 20∶1, 10∶1, and 5∶1. The cytotoxicity percentage was calculated as the percentage of specific ⁵¹Cr release, as described in the materials and methods section. Data are presented as mean ± standard error of the mean from triplicate samples of 11 repeated assays, each including 1 fed and 1 fasted mouse. (C) Histograms show the phenotype of Hepa1-6 cells that was analyzed using antibodies against mouse Fas, death receptor 5 (DR5), and decoy TRAIL receptor 1 and 2 (DcR1 and DcR2) in solid lines. Negative controls, which were stained with isotype-math antibodies, are indicated using dotted lines. The proportion of Hepa1-6 cells positive for those markers is provided. (D) Liver lymphocytes that were obtained from 3-day-fasted mice were incubated with CMA, anti-TRAIL mAb, anti-FasL mAb, or their combination before incubation with ⁵¹Cr-labeled-Hepa1-6 for 4 hours, at a lymphocyte: Hepa1-6 ratio of 40∶1. Bar graph shows the mean cytotoxicity percentage plus standard deviation for each group. Statistical analysis was performed for each ratio using the independent samples T test; *p <0.05.