Assays of Cell Death

At the close of an immune response, most of the activated immune cells die. Furthermore, the outcome of many immune responses is the death of infected or affected cells, and so immunologists utilize a battery of methodologies to test for cell death.

The ⁵¹Cr Release Assay Was the First Assay Used to Measure Cell Death

The ⁵¹Cr release assay was, for decades, the method of choice for measuring cytotoxic T cell– and natural killer cell–mediated killing and was used in the Nobel Prize–winning experiments performed by Doherty and Zinkernagel that first described the phenomenon of MHC restriction in T-cell recognition. Target cells are first incubated in a solution of ⁵¹Cr-labeled sodium chromate, which is taken up into the cells. Excess chromium is washed out of the cell suspension and the radioactively labeled targets are mixed with the killer-cell population at defined effector-to-target cell ratios. Death of the target cells is indicated by the release of ⁵¹Cr into the supernatant of the mixed cell culture, and is quantified by comparing the ⁵¹Cr release from the test cells with that from detergent-treated (maximum release control) and untreated (minimal release control) cells. ⁵¹Cr is a γ-emitting radioactive isotope, and the radioactivity in the assay supernatants can therefore be readily measured with a γ counter. A modern alternative to this technique uses CFSE to label the cells and quantifies the release of fluorescent material into the supernatant with a fluorescence plate reader. These two assays measure all forms of cell death.

Key Concept:

• In the ⁵¹Cr release assay, ⁵¹Cr is released from prelabeled cells on cell death. ⁵¹Cr release can therefore be used to measure the extent of cell death within the labeled population. Modern versions of this assay use fluorescent, rather than radioactive, reagents that are released into the supernatant on cell death.

Fluorescently Labeled Annexin A5 Measures Phosphatidylserine in the Outer Lipid Envelope of Apoptotic Cells

In cells undergoing apoptosis (programmed cell death), but not other modes of cell death, the membrane phospholipid phosphatidylserine flips from the interior to the exterior side of the plasma membrane phospholipid bilayer. Annexin A5 is a protein that binds to phosphatidylserine in a calcium-dependent manner; fluorescently labeled annexin A5 can therefore be used to tag apoptotic cells for detection using immunofluorescence microscopy, flow cytometry, or a fluorescence plate reader.

Key Concept:

• Fluorescently labeled annexin A5 binds to phosphatidylserine exposed on the surface of cells undergoing apoptosis and can therefore be used as a measure of apoptosis in immunofluorescence or flow cytometric experiments.

The TUNEL Assay Measures Apoptotically Generated DNA Fragmentation

Another common method for the detection of apoptotic cell death uses the fact that apoptotic cells undergo a process of progressive DNA degradation, which results in the generation of short DNA fragments within the nucleus. The TUNEL assay relies on the use of the enzyme terminal deoxynucleotidyl transferase (TdT) (see Chapter 6 for a detailed explanation of this enzyme’s activity) to add bases onto the broken ends of DNA sequences in a nontemplated manner. The classic variation of the TUNEL method uses TdT to add BrdU (see Figure 20-29) to fixed and permeabilized cells. BrdU is incorporated into the newly synthesized DNA, and is then detected with fluorescently labeled anti-BrdU antibodies. More recent iterations of this method use the incorporation of short DNA segments prelabeled with a molecule that then binds to a fluorescent tag under very gentle conditions.

FIGURE 20-29 Assessment of apoptosis, using a TUNEL assay. (a) Apoptosis results in DNA fragmentation by intracellular nucleases. (b) BrdU-labeled nucleotide triphosphates are added onto the broken ends of fragmented DNA in fixed and permeabilized apoptotic cells, using the enzyme TdT. (c) Fluoresceinated antibodies specific for BrdU can then be used to detect apoptotic cells.

The illustration shows a double helical strand of DNA with breaks in three regions identified by yellow stars. Text below reads, DNA strand breaks caused by endonucleases produced by apoptosis process. T d T and B r d U T P are added to the fragmented ends of the DNA. The illustration shows B r d U T P s bound to the 3 dash end of the DNA strand. Text below reads, Add B r d UTPs to 3¬-OH DNA strand breaks using Td T enzyme as catalyst. In the next step, Anti-B r d U F I T C labeling is done. The illustration shows labeled antibodies bound to B r d U T P that are bound to the 3 dash end of DNA. Text below reads, Fluoresceinated antibody labeling of B r d U T P attached to 3¬-OH DNA strand breaks.

Key Concept:

• Apoptotic cells undergo DNA fragmentation. The TUNEL assay uses terminal deoxynucleotidyltransferase to add labeled nucleotides onto the DNA ends exposed on apoptotic fragmentation. The amount of added label is a measure of the extent of apoptosis.

Caspase Assays Measure the Activity of Enzymes Involved in Apoptosis

Caspases are a family of cysteine proteases that cleave proteins after aspartic acid residues. Different members of the caspase family are activated during apoptotic cascades, depending on how the cascades are initiated. For example, caspase-8 is activated on engagement of the Fas receptor by Fas ligand. Several different types of caspase assays are now commercially available, including caspase detection kits that yield fluorescent products on caspase-mediated cleavage, as well as kits that detect the cleaved and active forms of caspases using Western blot methodology.

Key Concept:

• Caspases are activated in apoptotic cells, and fluorescence- or Western blot–based caspase assays therefore provide a measure of apoptosis.