Apoptosis and Cell Proliferation (522915), страница 13
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FluoresceindUTP) or with a secondary detection system which has a reportermolecule (e.g. fluorescein, AP, POD).P Advantages like in situ nick translation; in addition:P More sensitive: maximum intensity of labeling (cell staining) ofapoptotic cells is higher compared to ISNTP Fast kinetics of dUTP incorporation in comparison with the DNApolymerase method: 30 min is sufficient for the labeling reactionP Higher sensitivity for apoptosis: TUNEL preferentially labels apoptosisin comparison to necrosisP Direct detection possible by the use of Fluorescein-dUTP (withoutsecondary detection system) for maximum sensitivity and minimalbackgroundP With the direct TUNEL labeling assay less working steps are requiredthan with the indirect assay.P Labor-intensive and time-consuming; only a few tests may beperformed simultaneouslyP Many cells (2–5 x 106/test) requiredP Undefined cell loss during fixation procedure (loss of specific cellpopulation?)pages 27,29 of thisguideDetection of phosphatidylserine onsurface of apoptotic cellsP Apoptotic cells are incubated with an assay protein (e.g.
annexin V)conjugated to a reporter molecule.P Assay protein binds a membrane component (e.g. phosphatidylserine) found on the outer surface of apoptotic cells only.P A DNA strain is added to distinguish necrotic cells (permeable) fromapoptotic cells (impermeable).P Apoptotic cells are made visible by assay of reporter molecule in flowcytometer or under a microscope.P Unique marker for apoptosis related plasma membrane changesP Allows analysis by flow cytometry fluorescence microscopy, or lightmicroscopyP Allows simultaneous labeling of other cell surface antigensP Annexin-V-Biotin allows fixation following Annexin-V binding forfurther analysis of additional cellular parametersP Not specific for apoptosis: Annexin V can stain inner membrane ofruptured cells; must distinguish apoptotic from necrotic cells with anadditional DNA stainP Many cells (106/test) requiredP Cannot be used on tissue sections or any fixed samplespages 32,34 of thisguideTrypan Blue Exclusion AssayDamage/leakage of plasmamembraneP Cells are incubated with dye.P Dead cells take up dye; living cells do not.P Stained (dead or damaged) cells are determined under a lightmicroscope.P Dye binds to intracellular proteins of leaky cells.P The classical standard method to distinguish viable from dead cellsby light microscopyP Quick and cheapP Only a small fraction of total cells from a cell population is requiredP Each individual sample has to be counted: only a few tests may beperformed simultaneouslyP Subjective evaluationP Not suitable for detection of apoptosisP Stains only necrotic cells or very late apoptotic cells (secondarynecrosis)Propidium Iodide Exclusion AssayDamage/leakage of plasmamembraneP Cells are incubated with fluorescent dye.P Dead cells take up dye; living cells do not.P Stained (dead or damaged) cells are determined under a microscopeor by flow cytometry.P Dye binds to DNA of leaky cells.P The standard method to distinguish viable from dead cells by fluores- P Each individual sample has to be counted: only a few tests may becence microscopy and flow cytometryperformed simultaneouslyP Double labeling procedures possible: simultaneous detection of e.g.
P Not specific for apoptosissurface antigensP Quick and cheapP Only a small fraction of total cells from a cell population is requiredDAPI, ethidium bromide, propidium iodide*In Situ Cell Death Kit, Fluorescein, AP, PODDetection of translocated membranecomponent27Annexin V-FLUOSAnnexin V-BiotinAnnexin V-Staining KitAnnexin V-Alexa™ 568Propidium iodide solution*For productinformation, seepage 36of this guide*Sold only in the USG Table 11: Methods for studying apoptosis in individual cells.3839Cell Death – Apoptosis and NecrosisCell Death – Apoptosis and Necrosis1Apoptosis Assay MethodsApoptosis Assay Methods1Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsDetection of apoptosis-related proteins1.2.3 Detection of apoptosis-relatedproteinsThere are a number of genes that regulateapoptosis.
That is, the products of thesegenes interfere with apoptotic pathways.Assays to detect the proteins encoded bythese genes can complement the assays described in the previous sections.The study of apoptosis-regulating genesand gene products is still evolving. The picture so far is complex (as summarized inSection 1.1.3 of this guide). For instance, insome cases, the same gene has an effect onboth the survival of normal cells and thedevelopment of cancers by preventingapoptosis36. A detailed discussion of thefield is beyond the scope of this guide, butis covered in a number of reviews36, 37. Asan introduction to the field, we discuss thecharacteristics of a few of these apoptosisregulating proteins.1The relationship of the ced (caenorhabditiselegans cell death) genes to apoptosis in thenematode Caenorhabditis elegans has beenextensively studied38. Of these, the ced-3and ced-4 genes39 encode proteins thatmust be active to initiate apoptosis.
In contrast, the ced-9 gene product protects cellsfrom apoptotic cell death, ensuring theirsurvival40. In other words, apoptosisis more likely when levels of ced-3 andced-4 protein are high or when levels ofced-9 protein are low.In mammalian systems, the Bcl-2 protooncogene serves much the same functionas ced-9, blocking the induction of celldeath41. The Bcl-2 oncoprotein also protects against the cytotoxic effects of certaindrugs42.The Bcl-2 protein can dimerize with itselfor with the product of the bax gene43. Thepresence of the bax protein seems to counteract the anti-apoptotic activity of Bcl-2.In summary, apoptosis is more likely whenbax protein levels are high or when Bcl-2protein levels are low.40Another mammalian gene product, p53, is atumor suppressor because it induces apoptosis in potentially malignant cells44. Absence or mutation of the p53 gene productled to malignant transformation and immortalization of the cell.Increases in expression of a growth stimulating gene, the c-myc proto-oncogene, actually induces apoptosis in the absence ofother growth factors45, 46.
High levels ofthe Bcl-2 protein can counteract the effectof the c-myc protein.For the analysis of apoptosis-regulatingproteins, Roche Molecular Biochemicalsoffers a set of antibodies to p53 (and anELISA kit for the detection of p53 in fluidsor extracts), and an antibody to the Bcl-2oncoprotein.Cell surface receptor genes (APO-1/Fas/CD95), other growth-stimulating genes(e.g., Ras), and other tumor-suppressinggenes (e.g., Rb) have also been implicated inthe regulation of apoptosis2, 37. The Fas(CD95/APO-1) molecule has originallybeen identified as a cell surface receptorthat could mediate apoptotic cell death oftransformed cells and cause regression ofexperimental tumors growing in nudemice. The function of Fas was assessed byestablishment of mouse cell transformantsthat constitutively expressed human Fas.When the transformed cells were treatedwith the antibody to human Fas, the cellsdied by apoptosis within 5 hours, which indicated that Fas can transduce an apoptoticsignal and that anti-Fas works as an agonist.
The subsequent purification of humanAPO-1 antigen and molecular cloning ofits cDNA established the identity of APO-1and Fas. Meanwhile, numerous reportshave shown a pivotal role of Fas in variousphysiological and pathological processes.The Anti-Fas provided by Roche Molecular Biochemicals is suitable for the induction of apoptosis as well as for the detectionof the Fas receptor.Anti-Fas (CD95/Apo-1)Cat.
No. 1 922 432100 µgTypeMonoclonal antibody, clone 2R2, IgG3, mouseUseful forApoptosis induction in Fas expressing cellsSamplesCell suspensions, adherent cellsMethodDirect induction of apoptosis by adding antibody to cell culturesTimeApprox. 3–5 h (induction of apoptosis)Significance of reagent: The antibodymay be used for the induction of apoptosisin cell cultures through Fas by imitating theFas-ligand. The Fas (CD95/Apo-1) molecule has been identified as a cell surfacereceptor that could induce apoptotic celldeath of transformed cells upon activationby its ligand and cause regression of experimental tumors in mice.Test principle: The antibody may be usedfor induction of apoptosis:Add antibody (1 µg/ml) into culturemedium of Fas-bearing cells2Incubation for 3–5 hoursSpecificity: The antibody was generatedby immunizing mice with transformed murine L-cells bearing recombinant humanFas receptor.
On Western blots, Anti-Fasbinds the human Fas/Apo-1 (CD95).3Detection of apoptosis by various assaysCan be used for:Antibody supplied as: Mouse monoclonalantibody (clone 2R2, IgG3) in cell culturesupernatant; sterile filtered.1Sensitivity: The antibody is suitable forinduction of apoptosis at 0.5 µg/ml inSKW6.4 and Jurkat cells. If secondary crosslinking with an anti-mouse IgG is used,the apoptosis inducing concentration couldbe reduced to 100 ng/ml. In Fas transfectants apoptosis is induced without crosslinking at 100 ng/ml. It has to be mentioned, that some Jurkat subclones do notor only in high doses respond to Anti-Fasinduction of apoptosis.1Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsDetection of apoptosis-related proteinsP Induction of apoptosis through the Fasreceptor41Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsDetection of apoptosis-related proteinsAnti-Fas-Biotin (CD95/Apo-1)Cat.
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