Figure 8.

Interferon (IFN)-γ in combination with TNF-α induces cell death in mouse lung carcinoma cells. L929 fibroblasts and Lewis lung carcinoma (LLC) cancer cells were cultured for 24 h before starting treatment with recombinant cytokines. Both L929 and LLC cells were (A,B) left untreated for 48 h and used as negative controls or (C) L929 cells were treated with 50 ng/mL TNF-α for 48 h and used as a positive control. LLC cell viability was retained after treatment with (D) 50 ng/mL TNF-α or (E) 100 ng/mL IFN-γ and after (F) simultaneous treatment with both 50 ng/mL TNF-α and 100 ng/mL IFN-γ for 24 h. Cell death was induced in LLC cells after (G) simultaneous treatment with 100 ng/mL of IFN-γ in combination with 50 ng/mL of TNF-α for 48 h and after (H) LLC pretreatment with 100 ng/mL of IFN-γ for 24 h followed by treatment with TNF-α for 24 h. The resulting cell monolayers were analyzed using bright-field microscopy (scale bar, 50 µM). Cell death was determined using flow cytometry analysis after cell staining with annexin V-FITC (depicted on the x-axis) and propidium iodide (PI, depicted on the y-axis). Annexin V-positive/PI-negative cells were regarded as apoptotic, whereas annexin V-positive/PI-positive cells were regarded as necrotic. Experiment was performed in duplicates and repeated two times with standard error not exceeding 10% between independent experiments. One representative experiment is shown, where the percentages of the four distinct cell populations are averages of duplicates with SEM <5%.