Apoptosis and Cell Proliferation (522915), страница 28
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The percentage of necroticcells is determined by trypan blue staining.The package insert for our ApoptoticDNA-Ladder Kit, Cat. No. 1 835 246, describes the purification of nucleic acidsfrom whole blood and cultured cells. Byfollowing the modified procedure decribedhere it is also possible to use tissue samples.Preparation of solutionsP HEPES buffer: Prepare according to theinstructions in the Annexin-V-Biotinpack insert.All steps can be performed at room temperature1Incubate 1 x 106 cells in 100 µl Annexin-V-Biotin working solution for10–15 min.2Wash 2 times with HEPES buffer.For suspension cells: Continue withstep 3.For adherent cells: Continue withstep 4.3Resuspend suspension cells in 1 mlHEPES buffer. Transfer 2 x 105 cells toa slide using cytospin device.4Air dry cells.
Fix with methanol/ethanol1:1 for 90 sec.5Air dry cells. Add 100 µl StreptavidinPOD (Cat. No. 1 089 153) working solution, incubate for 1 h.6Rinse with HEPES buffer7Add DAB substrate solution (Cat. No.1 718 096) working solution, incubatefor 10–15 min.8Rinse with HEPES buffer9Analyze samples under a light microscope.Preliminary InformationP Weight of sample: The tissue sampleshould weigh between 25 and 50 mg.P Additional required solutions:– Lysis buffer: Prior to extraction ofDNA, prepare a lysis buffer. 200 µlof this buffer are sufficient for onetissue sample.
The lysis buffer consists of 4 M urea, 100 mM Tris,20 mM NaCl and 200 mM EDTA,pH 7.4 (25°C).– Proteinase K solution: 20 mg/ml in50 mM Tris-HCl (pH 8.0) and 1 mMCaCl2.Protocol for isolation of DNA from tissue samples1 Add 200 µl lysis buffer and 40 µl proteinase K solution to 25–50 mg tissue,mix.2 Incubate for 1 h at 55°C.3 Add 200 µl binding buffer, mix.4 Incubate for 10 min at 72°C.5 Proceed with the addition of 100 µl isopropanol as described in the pack insert(3rd.
step of section 5).Note: Be aware, that apoptosis is a single cellevent, and therefore in most tissues you willnot find a sufficient number of apoptoticcells to produce a DNA ladder.AppendixP Annexin-V-Biotin working solution:Dilute 20 µl. Annexin-V-Biotin labelingreagent in 1000 µl incubation buffer(sufficient for 10 samples).1093Technical tipsTechnical tips on the Cell Proliferation ELISA kits3.1.5 Technical tips on the CellProliferation ELISA kitsHow to interrupt the proliferation assayThe detection of BrdU-labeled DNA withthe Cell Proliferation ELISAs does nottake more than 1.5–3 hours. Nevertheless,the labeling period which may vary between 2 and 24 hours can get the scientist intime trouble.
Our assay can be interruptedafter the labeling process: After the removalof the culture medium, the protocol proceeds with the drying of the labeled cellsusing e.g. a hair-dryer. The dry cells staysafe and sound up to one week when storedat 4°C in the microtiter plate before theyare fixed and denatured according to theprovided protocol.AppendixA tip for measuring lymphocyte proliferationTo study the proliferation of lymphocytes,the cells are stimulated e.g. with growthfactors, cytokines or mitogens.
The increase in cell numbers can (in special cases)lead to cluster formation of the lymphocytes: Cells from the same progenitor sticktogether and form aggregates in the cultureplate.This effect may disturb the antibodyrecognition of the ELISA system andthereby result in an underestimation of response. To avoid signal variation: Carefullyresuspend the cells after the BrdU-labelingperiod and before centrifugation for removing the culture medium. This will enablethe equal accessibility of each cell for theantibody recognizing the BrdU-label.3110Special applications of cell death and cell proliferation methodsTUNEL assays3.2 Special applications of cell death and cellproliferation methods3.2.1 TUNEL assays3.2.1.1 Discrimination between dead andviable apoptotic cells using two-color TdTassay and surface labeling as detectedby flow cytometry[from Earl A. Timm, Jr.
and Carleton C. Stewart,Laboratory of Flow Cytometry, Roswell Park CancerInstitute, Buffalo, N.Y., USA]Note: This article appeared in BiochemicaNo. 1 (1996), 44–47.Summary: The TUNEL method usesterminal dideoxynucleotidyl transferase(TdT) to incorporate hapten-tagged nucleotides into the 3’-strand breaks that occur in DNA during apoptosis (Gorczyca etal., 1993; Chapman et al., 1995). If these nucleotides are coupled to a fluorescent molecule, or if the hapten can be detected by afluorescent secondary reagent, the apoptotic cells can be analyzed by flow cytometry.Flow cytometry permits not only the detection of apoptotic populations, but alsothe simultaneous detection and immunophenotyping of necrotic populations.
Thedrawback to using the current TdT method, however, is that the ethanol permeabilization of the cells is incompatible withimmunophenotyping because it denaturescellular epitopes (Darzynkiewicz et al.,1992; Li et al., 1995).A protocol has been developed that bothpreserves the surface markers and detectsapoptotic cells.
In addition, it is possible todiscriminate between dead apoptotic cellsand viable apoptotic cells with a secondhapten-tagged nucleotide that labels deadcells. The method also can distinguish deadapoptotic cells from cells that have died byother mechanisms (e.g., necrosis).3.2.1.2 The use of flow cytometryfor concomitant detection of apoptosisand cell cycle analysis[from E. Hanon, A. Vanderplasschen and P.-P. Pastoret,Department of Immunology/Vaccinology,Faculty of Veterinary Medecine, University of Liège,Liège, Belgium]Note: This article appeared in BiochemicaNo. 2 (1996), 25–27.Summary: Two distinct modes of celldeath, apoptosis and necrosis, can be distinguished on the basis of differences inmorphological, biochemical, and molecularchanges occuring in the dying cells (Duvalland Wyllie, 1986).Cells undergoing apoptosis display a characteristic pattern of structural changes inthe nucleus and cytoplasm, including rapidblebbing of the plasma membrane andnuclear disintegration (Duvall and Wyllie,1986).
Extensive damage to chromatin andcleavage of DNA into oligonucleosomallength fragments both occur during apoptosis (Duvall and Wyllie, 1986).Several flow cytometric methods for identifying cells undergoing DNA fragmentation have been described recently.
Theseinclude DNA content analysis and in situlabeling of DNA fragments with tracerdUTP. The former is based on the accumulation of ethanol-fixed apoptotic cells in thesub-G0/G1 peak of DNA content histogram as a result of loss of DNA fragmentsout of the cells and because of a reducedDNA “stainability” (Telford et al.,1991,1992). The latter uses exogenous terminaldeoxynucleotidyl transferase (TdT) to labelin situ the DNA strand breaks with a tracer-dUTP (Gorczyca et al., 1993; Sgonc etal., 1994).Recent observations have revealed a profound regulatory interrelationship betweenapoptosis and the cell cycle (Gorczyca etal.). The investigation of this relationshipideally requires techniques that permit concomitant apoptosis detection and cell cycleanalysis at a single-cell level.AppendixThis section of the Appendix contains condensed versions of articles that appearedin the Boehringer Mannheim Biochemicanewsletter.
For further experimental detailand background, see the full Biochemicaarticles.1113Special applications of cell death and cell proliferation methodsTUNEL assaysTwo flow cytometric techniques are usually used to investigate the cell cycle: DNAquantification to identify the cell cycle position (Vindelov et al., 1990) and detectionof bromodeoxyuridine (BrdU) incorporation to reveal cells going through the Sphase (Gratzner, 1982). In this investigation, the development of flow cytometrictechniques that permit concomitant detection of apoptosis and cellular DNA contentor BrdU content analysis by adapting theapoptosis detection protocol of the Boehringer Mannheim In Situ Cell Death Detection Kit, Fluorescein is reported.3.2.1.3 Comparison of two cell deathdetection methods: In situ nick translation and TUNEL[from Maria Pihlgren, Joelle Thomas, and JaquelineMarvel, Immunologie cellulaire, Lyon Cedex, France]Note: This article appeared in BiochemicaNo.
3 (1996), 12–14.Summary: Apoptosis is a form of regulated cell death characterized by specificmorphological changes. These include cellshrinkage, membrane blebbing, chromatincondensation, and cell fragmentation intosmall apoptotic bodies. At the molecularlevel, the activation of an endogenous endonuclease results in the fragmentation ofcellular DNA into oligosomal length fragments (Martin et al.
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