Once lymphocyte culture had been optimized using the proliferative response to polyclonal mitogens as an assay, it became possible to detect antigen-specific T-cell proliferation in culture by measuring 3H-thymidine uptake in response to an antigen to which the T-cell donor had previously been immunized (Fig. A.34). This is the assay most commonly used for assessing T-cell responses after immunization, but it reveals little about the functional capabilities of the responding T cells. These must be ascertained by functional assays, as outlined in Sections A-28 and A-29.
A-27 Measurements of apoptosis.
Apoptotic cells can be detected by a procedure known as TUNEL (TdTdependent dUTP–biotin nick end labeling) staining. In this technique, the 3ʹends of the DNA fragments generated in apoptotic cells are labeled with biotin-coupled uridine by using the enzyme terminal deoxynucleotidyl transferase (TdT). The biotin label is then detected with enzyme-tagged streptavidin, which binds to biotin. When the colorless substrate of the enzyme is added to a tissue section or cell culture, it produces a colored precipitate only in cells that have undergone apoptosis (Fig. A.35).
Additional methods are often used to detect apoptosis of cells in experimental animals. One simple method is to incubate cells with a fluorescently labeled preparation of the protein Annexin V. This protein has a high affinity for a specific membrane phospholipid, phosphatidylserine (PS). In healthy cells, PS is found exclusively on the inner leaflet of the plasma membrane, and is therefore inaccessible to extracellular incubation with Annexin V. When cells are undergoing apoptosis, the PS is transported to the outer cell surface, where it can be bound by the fluorescently labeled Annexin V, which is then detected
Fig. A.34 Antigen-specific T-cell proliferation is used frequently as an assay for T-cell responses. T cells from mice or humans that have been immunized with an antigen (A) proliferate when they are exposed to antigen A and antigen-presenting cells but not when cultured with unrelated antigens to which the hosts have not been immunized (antigen B). Proliferation can be measured by incorporation of 3H-thymidine into the DNA of actively dividing cells. Antigen-specific proliferation is a hallmark of specific CD4 T-cell immunity.
Fig. A.35 In the TUNEL assay, fragmented DNA is labeled are added (second panel). The biotinylated DNA can be detected by by terminal deoxynucleotidyl transferase (TdT) to reveal using streptavidin, which binds to biotin, coupled to enzymes that apoptotic cells. When cells undergo programmed cell death, or convert a colorless substrate into a colored insoluble product (third apoptosis, their DNA becomes fragmented (first panel). The enzyme panel). Cells stained in this way can be detected by light microscopy, TdT is able to add nucleotides to the ends of DNA fragments; most as shown in the photograph of apoptotic cells (stained red) in the commonly in this assay, biotin-labeled nucleotides (usually dUTP) thymic cortex. Photograph courtesy of R. Budd and J. Russell.