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[moljul]

during my recherche about proliferation/viability/apoptosis assay I stumbeled upon the following experimental setting:

the cytotoxic effects of human tumor necrosis factor alpha (hTNF- on the mouse fibrosarcoma cell line WEHI-164 should be tested:

Cells in culture (106 cells/ml) were preincubated with actinomycin C (1 μg/ml) for 3 h. Aliquots of these pretreated cells were transferred to a microtiter plate (5 x 104 cells/well) and incubated with actinomycin C and various amounts of hTNF-alpha for 24 h. Cellular response to TNF was
measured with the Roche Molecular Biochemicals Cell Proliferation Kit II (XTT) and plotted against TNF concentration.


I wondering, why cells are pretreated with actinomycin C prior to TNFa exposure! Are cytotoxicity assays are always done like this, or is it possible only to treat cells with TNFa, to test its cytotoxic effect??

thanks for recommendation

[Inmost sun]

moljul, on May 21 2010, 09:36 AM, said:

during my recherche about proliferation/viability/apoptosis assay I stumbeled upon the following experimental setting:

the cytotoxic effects of human tumor necrosis factor alpha (hTNF- on the mouse fibrosarcoma cell line WEHI-164 should be tested:

Cells in culture (106 cells/ml) were preincubated with actinomycin C (1 μg/ml) for 3 h. Aliquots of these pretreated cells were transferred to a microtiter plate (5 x 104 cells/well) and incubated with actinomycin C and various amounts of hTNF-alpha for 24 h. Cellular response to TNF was
measured with the Roche Molecular Biochemicals Cell Proliferation Kit II (XTT) and plotted against TNF concentration.


I wondering, why cells are pretreated with actinomycin C prior to TNFa exposure! Are cytotoxicity assays are always done like this, or is it possible only to treat cells with TNFa, to test its cytotoxic effect??

thanks for recommendation

not necessarily; the experiment described must be seen in the light of the aim of the study which you do not describe; nevertheless, you can, of course, test TNF alone