Analyzing Apoptosis and Necrosis Data
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You are working in a laboratory investigating the effects of new chemicals called X and Y that kill cells. To test whether X and Y cause necrosis or apoptosis, you briefly treat cultured cells with each chemical and perform several assays.
A. First, you examine the chromosomal DNA. You isolate DNA from cells, label 5' ends with radioactive ATP by using polynucleotide kinase, run the DNA on a gel, and detect radiolabeled fragments. Your results are shown in Figure A . The lane from the untreated control cells is labeled U. Does X cause apoptosis? What about Y? Explain the origin of the ladder in the Y lane.
B. Second, you examine the subcellular localization of cytochrome c using immunofluorescence microscopy. Unfortunately, during sample preparation you mixed up the samples. Assign a likely identity (U, X, or Y) to the samples labeled 1, 2, and 3 in Figure B.
C. Third, without making the cells permeable in any way, you stain them with Annexin V linked to a green fluorophore, which binds phosphatidylserine, and propidium iodide, which fluoresces red when it binds DNA. Neither of these compounds can travel across intact plasma membranes. Again during sample preparation you mixed up the samples. Assign a likely identity to the samples in Figure C. Annexin V staining is shown as \ hatching, propidium iodide staining is shown as // hatching, and staining with both in the same region appears as crosshatching.
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Solution Summary
In this solution, we take a look at analyzing chromosomal and cellular data regarding death. We are testing a compound designed to kill cells, and we need to know what kind of death (apoptosis or necrosis) is occurring. How do we figure this out? Download the solution to find out!
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A) The extent of chromosomal damage is far less in the X-treated compared to the Y. The X DNA runs basically identical to the untreated control, suggesting it is not causing apoptosis. Y, on the other hand, seems to have an effect. This laddering is due to fragmentation of the chromosome that occurs during apoptosis by restriction enzymes. We know this is a ...
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