Apoptosis and Cell Proliferation (522915), страница 29
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1994). These can bereadily detected by DNA gel electrophoresis. However, gel electrophoresis does notallow the detection of apoptosis in individual cells.AppendixIn contrast, techniques that use enzymaticlabeling of DNA strand breaks can provideinformation regarding apoptosis at a singlecell level. The TdT-mediated dUTP NickEnd Labeling (TUNEL) technique usesterminal deoxnucleotidyl transferase (TdT)and allows the labeling of double-strandedDNA breaks (free 3’-OH DNA ends),while the In Situ Nick Translation (ISNT)method employs DNA Polymerase I anddetects single-stranded DNA breaks.Another advantage of these techniques isthat they can be used in combination withcell surface staining or cell cycle analysis.The abilities of the TUNEL and ISNTtechniques to detect apoptosis in two typesof cells: the IL-3-dependent cell line BAF3 and freshly isolated CD8+ lymphocytesfrom mouse spleen are compared.31123.2.1.4 Fixation of tissue sectionsfor TUNEL combined with stainingfor thymic epithelial cell marker[from Olav Schreurs, Trond S.
Halstensen, ZlatkoDembic, Bjarne Bogen, Karl Schenck, Department ofOral Biology, University of Oslo, Oslo, Norway]Note: This article appeared in BiochemicaNo. 4 (1997), 19–21.Summary: In the thymus, positive andnegative selection of thymocytes are important forces that shape the repertoire ofmature T lymphocytes in the immune system. In studies on negative selection, it is ofgreat interest to determine whether apoptotic cells reside in thymic cortex or medulla (Surh et al. 1994; Kisielow et al.
1995).Terminal dUTP nick end labeling(TUNEL) is a technique, that is well suitedto demonstrate apoptosis in situ, and themethod may be combined with labeling ofother markers. In order to distinguish between thymic cortex and medulla, differential expression of cytokeratin, MHC classII molecules and epithelial cell markershave been used (Surh et al.
1994; Wack et al.1996; Douek et al. 1996). In the course of aninvestigation on deletion of tumor-specificTCR-transgenic T-cells in the thymus(Lauritzsen et al.), TUNEL has been combined with commercially available monoclonal antibodies, that are monospecific forthymic epithelial cells, to unambiguouslylocalize T-cell deletion.
During the courseof these studies, we established fixatingconditions, that gave us superior results.Special applications of cell death and cell proliferation methodsMetabolic assays, Annexin assays3.2.2 Metabolic assays, Annexin assays3.2.3 Annexin assays3.2.2.1 Biochemical and cellular basisof cell proliferation assays that usetetrazolium salts3.2.3.1 The use of annexin forconcomitant detection of apoptosis andcellular phenotype[from Michael V. Berridge, An S. Tan, Kathy D. McCoy,and Rui Wang, Malaghan Institute of Medical Research,Wellington School of Medicine, Wellington South, NewZealand][from S. Hoornaert, E. Hanon, J. Lyaku, and P.-P. Pastoret, Department of Immunology/Vaccinology, Faculty ofVeternary Medicine, University of Liège, Liège, Belgium]Summary: Tetrazolium salts (such asMTT, XTT, and WST-1) are used extensively in cell proliferation and cytotoxicityassays, enzyme assays, histochemical procedures, and bacteriological screening.
Ineach, these tetrazolium salts are metabolically reduced to highly colored end products called formazans. Yet, the nature oftheir cellular bioreduction is poorly understood despite their long-time use (Stowardand Pearse, 1991).In our laboratory, we demonstrated thatmost cellular reduction of MTT was dependent on the reduced pyridine nucleotides NADH and NADPH, not onsuccinate as had been previously believed(Berridge et al., 1993, 1994; Berridge andTan, 1993).
Cellular reduction of MTT wasassociated with enzymes of the endoplasmic reticulum and was more relatedto NADH production through glycolysisthan to respiration.Recently, assays have been introducedbased on tetrazolium salts (such as XTTand WST-1) that are reduced to solubleformazans. These assays depend on intermediate electron acceptors such as phenazine methosulfate (PMS).The question arises: Is the cellular reduction of these new salts similar to that ofMTT? In this article, the answer to thatquestion is attempted.In summary, it could be shown that, unlikeMTT, XTT and WST-1 are efficiently reduced by NADH and NADPH in the absence of cells or enzymes, and their reduction involves superoxide.
Cellular reduction of WST-1 occurs at the cell surface andalso involves superoxide.Note: This article appeared in BiochemicaNo. 3 (1997), 19–20.Summary: Two distinct modes of celldeath, apoptosis and necrosis, can be distinguished on the basis of differences inmorphological and biochemical characteristics. Under the elctron microscope, cellsundergoing apoptosis display cell shrinkage, apoptotic body formation, and chromatin condensation. Biochemically, theapoptotic process is charaterized by fragmentation of DNA into oligonucleosomalfragments.
Furthermore, during the earlystages of apoptosis, changes also occur atthe cell surface membrane (Andree et al.1990; Creutz, 1992; Fadok et al. 1992). Oneof these plasma membrane alterations is thetranslocation of phosphatidylserine (PS)from the inner part to the outer layer of theplasma (Vermes et al., 1995), thus exposingPS at the external surface of apoptotic cells,where it can be specifically recognized bymacrophage (Fadok et al.
1992).Annexin V, a Ca2+-dependent phospholipid-binding protein, possesses high affinityfor PS (Vermes et al., 1995) and can thus beused for detecting early apoptotic cells(Koopman et al. 1992, Verhoven et al. 1995,Vermes et al. 1995, Homburg et al. 1995).Since annexin V can also detect necroticcells as a result of the loss of membrane integrity, apoptotic cells have to be differentiated from these necrotic cells by the use ofpropidium iodide (PI). Indeed, PI selectively labels necrotic, but not apoptotic cells.Several studies have revealed a correlationbetween apoptosis and cell phenotype(Carbonari et al. 1995, Lewis, et al. 1994).The investigation of this relationship ideally requires techniques that permit the concomitant detection of apoptosis and cellphenotype analysis at a single cell level.
Inthis report, the development of a procedurewhich permits concomitant detection ofapoptosis and cell phenotype characterization by flow cytometry is described.AppendixNote: This article appeared in BiochemicaNo. 4 (1996), 14–19.1133Special applications of cell death and cell proliferation methodsBrdU assays3.2.4 BrdU assays3.2.4.1 Detection of bromodeoxyuridinein paraffin-embedded tissue sectionsusing microwave antigen retrievalis dependent on the mode of tissuefixation[from Wesley M. Garrett and H.D. Guthrie, Germplasmand Gamete Physiology, Agricultural Research Service,Beltsville, United States]Note: This article appeared in BiochemicaNo.
1 (1998), 17–20.AppendixSummary: A simple routine microwaveantigen retrieval procedure allows the sensitive detection of incorporated BrdU inpulse labeled cells. Of the two fixativestested, Carnoy’s offers superior nuclearmorphology, but with a sacrifice of immunostaining intensity. For investigationswhere animals are sacrificed within severalhours after pulse labeling, Carnoy’s fixativemay prove adequate for a general fixative,but it is not known what effect it has oncellular antigens of interest. For our purposes, 10% neutral buffered formalin wasfound to be a superior fixative, because ofits ability to cross-link nuclear proteins andassociated chromatin, resulting in more intense immunostaining for BrdU.
In addition, we have found that formalin fixationcoupled with microwave antigen retrieval iscompletely compatible with immunostaining of other antigens of interest.3114ReferencesApoptosis-related parameters3.3 References3.3.1 Apoptosis-related parameters – Abbreviations and ReferencesParameterFull length nameReferenceAIFApoptosis inducing factorP Susin S. A. et al. (1996) J. Exp. Med. 184, 1331.ApafApoptotic proteaseactivating factorP Zou, H.
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