We test tumors using at least 3 of 5 available assay endpoints. In the DISC (Differential Staining Cytotoxicity) assay, originated by Dr. Weisenthal while at the NCI, the entire contents of the cell culture are cytocentrifuged onto permanent microscope slides and differentially stained to allow discrimination of normal and neoplastic cells and living and dead cells. The endpoint for cell death is delayed loss of membrane integrity, which has been found to be a surrogate for apoptosis. Advantages of the DISC assay include direct visualization of tumor cells and establishment of a permanent archival record.  The DISC assay was the first of the new-generation functional tumor cell profiling methods to feature the cell death endpoint, upon which nearly all new-generation functional profiling assays subsequently were based.  Interpretation of DISC assay slides is highly labor-intensive but the assay is widely-regarded among experts in the field to be the gold standard owing to the ability examine directly each cell in order to positively discriminate tumor cells from non-tumor cells and to better characterize drug effects upon the entire tumor cell population.  Click here to see a simplified graphic DISC assay methodology flowchart. 

 

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay measures mitochondrial metabolism in the entire cell culture. In the assay, yellow tetrazolium salt (MTT) is reduced in metabolically active cells to form purple formazan.  The color can then be quantified by spectrophotometry, enabling an accurate measurement of metabolic activity.

 

The ATP (Adenosine Triphosphate) assay measures cellular ATP content by luminometry, based on the luciferin/luciferase reaction. Cells maintain a critical ATP thresholds whose measurement reflects cell viability, specifically indicating, in functional tumor cell profiling, whether apoptotic cell death has occurred during drug exposure.

 

The redox (resazurin) assay measures total metabolic activity in the entire cell culture, using the Alamar Blue reagent. 

 

The caspase 3/7 assay measures the activation of caspases 3 and 7 using luminometry.

 

All of the above are cell death endpoints.