Apoptosis and Cell Proliferation (522915), страница 26
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It may be used for studies in embryogenesis and tumor research.Anti-Ki-67 (Ki-S5)(clone Ki-S5)Formalin gradeIn Western blots the Ki-S5 antibody recognizes a protein of 345 kD and 395 kD identicalwith the Ki-67 antigen. The immunoreactivity of Ki-S5 is confined to the nuclei proliferating cells and no cross-reactivity with cytoplasmic antigens of epithelial occurs.
Acomparison of immuno-histochemical labeling of fresh and fixed tissue samples of NHLshowed that identical results were obtained with Ki-67 and Ki-S5.Anti-PCNA/Cyclin(clone 19F4)The antibody reacts with proliferating cell nuclear antigen (PCNA = an auxiliary proteinof DNA polymerase d), a polypeptide of 36 kD. Anti-PCNA is used to determine theproliferative cell fraction in various tumors.Anti-PCNA/CyclinFluorescein(clone 19F4)The antibody reacts with proliferating cell nuclear antigen (PCNA = an auxiliary proteinof DNA polymerase d), a polypetide of 36 kD. Anti-PCNA-Fluorescein is used for themeasurement of proliferating cells by flow cytometry.Anti-PCNA/Cyclin(clone PC10)Formalin gradeThe antibody reacts with proliferating cell nuclear antigen (PCNA = an auxiliary proteinof DNA polymerase d), a polypeptide of 36 kD.
Anti-PCNA is used to determine theproliferative cell fraction in various tumors.AntiTopoisomerase IIa(clone Ki-S1)Formalin gradeThe antibody recognizes a major protein of 170 kDA, the a isoform of topoisomerase II.It binds to the carboxyterminal a-isoenzyme specific epitope missing in topoisomeraseIIb. In immunohistochemistry the antibody shows strong nuclear staining also in paraffin-embedded tissue sections. It binds only to proliferating cells, while resting, non-cycling cells are not labeled.
This specificity for proliferating cells has allowed the antibodyto be used for determination of the proliferative fraction in solid tumors such as mammary carcinomas and gangliomas.Anti-TransferrinReceptor, human(clone B3/25)The antibody reacts with the human transferrin receptor glycoprotein. The transferrinreceptor participates in the uptake of tranferrin, the major serum iron transport protein.The transferrin receptor is present on all cells (except erythrocytes) but is especiallydense on the surface of rapidly proliferating cells. It can be used therefore, as a proliferation marker.G Table 20: Specificity of monoclonal antibodies to cell-cycle associated antigens.Methods for studying cell proliferation and viability in individual cellsAssays that monitor expression of cell cycle-associated antigensformalin-fixed, paraffin-embedded normal lymphatic tissue from tonsil.
The slide shows a secondary follicle with normal cell layering. Slide was counterstainedwith hematoxylin. (Data kindly provided by Dr. H. Merz).Result: Lymphoblasts are observed in the upper righthand corner of the follicle (darkly stained area). Thecortex, which is leukocyte-rich and lymphocyte-poor,is much less stained. However, lymphoblasts are seensporadically in the diffuse cortex.G Figure 70: Flow cytometric analysis, Anti-Topoisomerase II-alpha (clone Ki-S1) and propidium iodide staining of human peripheral blood lymphocytes. Human PBL were stimulated withphytohemagglutinin A.
At timed intervals, aliquots of the cell preparation were stained for cell cycleposition (with Anti-Topoisomerase II and Anti-mouse-Ig-fluorescein, according to the pack insert) andtotal DNA content (with propidium iodide, according to standard procedures). The histograms showthe two-parameter flow cytometric analysis of the cells at the time of stimulation (a = 0 h), and at24 h intervals after stimulation (b= 24 h, c = 48 h, d = 72 h).
The cell cycle phases are indicated onhistogram c. FITC-Fluorescein, intensity of Anti-Topoisomerase II staining; PI Fluorescence, intensityof propidium iodide staining.Result: Topoisomerase IIa can be found during G1, S, G2, and M phases in proliferating cells(histograms c-d), but is not expressed in resting (G0) cells (histogram a).Antibody to/Conjugated to(Clone)Antigen expressed duringG0G1SG2MAnti-Casein Kinase 2a (clone 1AD9)–++++1 499 602100Anti-PCNA/Cyclin (clone 19F4)–+++++1 170 406801 205 81140Anti-PCNA/Cyclin (clone PC10)–++++1 486 772100Anti-Ki-67 (clone Ki-S5)–++++1 742 3451000Anti-Topoisomerase IIa (clone Ki-S1)–++++1 742 3531000Anti-Transferrin-Receptor (clone B3/25)+++++1 118 048200Anti-PCNA/Cyclin-Fluorescein (clone 19F4)Cat. No.See aboveNo. oftests*F Table 21: Monoclonal antibodies tocell-cycle associated antigens* Flow cytometric assaysOther applications: For examples of howthe Anti-PCNA/Cyclin antibody may beused in the lab, see Appendix, page 126.99Cell Proliferation and ViabilityG Figure 69: Anti-Ki-67 (clone Ki-S5) staining of2Methods for studying cell proliferation and viability in all populationsMethods for studying cell proliferation and viability in all populationsSummary of methods for studying cell proliferation and viability in individual cellsSummary of methods for studying cell proliferation and viability in individual cells2.2.2.3 Summary of methods for studying cell proliferation and viability in individual cellsDNA SynthesisAssay principleAdvantagesLimitationsAutoradiographyP The samples are incubated with [3H]-TdR for a certain period of time.
If [3H]-TdR isP Quantitative detection of S phase cells: Determination of growingP Long exposure time (days) requiredP Radioactive isotope, handling and storage problemspresent for 1 h or less, only those cells which are in the S-phase (DNA synthesis) ofthe cell cycle will be labeled.P The samples are fixed and immersed in emulsion.P The radiolabel is visualized as black grains on the film.P The samples are incubated with BrdU for a certain period of time. If BrdU is presentP Quantitative detection of S-phase cells: Determination of growingBrdU Labeling and Detection Kit Ifor 1 h or less, only those cells which are in the S-phase (DNA synthesis) of the cellcycle will be labeled.P The samples are fixed and the DNA is denatured.P Incorporated BrdU is bound by a fluorescein-conjugated monoclonal antibodyagainst BrdU.P Bound Anti-BrdU-Fluorescein is detected by fluorescence microscopy or flowcytometry.fraction in populationP Results within a few hoursP Can counterstain the tissue simultaneously to reveal tissuemorphologyImmunocyto/histochemistry (light microscopy)P The samples are incubated with BrdU for a certain period of time.
If BrdU is presentP Quantitative detection of S-phase cells: Determination of growingfor 1 h or less, only those cells which are in the S-phase (DNA synthesis) of the cellcycle will be labeled.P The samples are fixed and the DNA is denatured.P Incorporated BrdU is bound by an alkaline phosphate (AP)-conjugated monoclonalantibody against BrdU.P Bound anti-BrdU AP is detected by a substrate reaction and visualized by lightmicroscopy.fraction in populationP Results within a few hoursP Can counterstain the tissue simultaneously to reveal tissuemorphologyImmunocytochemistry (fluorescence microscopy)Cell Proliferation and ViabilityIn Situ Cell Proliferation Kit, FLUOS2fraction in populationIn Situ Cell Proliferation Kit, APBrdU Labeling and Detection Kit IIP Stained samples cannot be stored for long periods of timeP Histological tissue organization cannot be observed simultaneouslyFor productinformation, seepages 87,88 of thisguidepages 87,90 of thisguideG Table 22: Summary of methods to study DNA synthesis in individual cells.Cell cycle-associated antigensMethod/Roche Molecular Biochemicals productAssay principleAdvantagesLimitationsImmunocytochemistry monoclonal antibodiesP The fixed and permeabilized cells are incubated with an antibody directed against aP No prelabeling of the cells required: each cell type/tissue may beP Detection of the antigen strongly depends on the fixation procedure: page 97cell cycle/proliferation-associated antigen (e.g., Ki-67, PCNA, Topoisomerase IIa).P The antibody bound to the intracellular antigen is detected by a fluoresceinconjugated anti-mouse Ig antibody.P Bound fluorescein-conjugated antibody is visualized by fluorescence microscopy ormeasured by flow cytometry.analyzedP Quantitative detection of the proliferative cell fractions (e.g., in solidtumors)P Results within a few hoursP Can counterstain the tissue simultaneously to reveal tissuemorphologyP The fixed tissue sections are incubated with an antibody directed against a cellSee aboveImmunohistochemistry monoclonal antibodiessome antibodies may not work on some tissue sections when theantigen is altered by the fixation step (e.g., formalin fixed paraffinembedded tissue sections)See aboveFor productinformation, seeof this guideSee abovecycle/proliferation-associated antigen (e.g., Ki-67, Topoisomerase IIa).P The antibody bound to the intracellular antigen is detected by an alkaline phosphatase (AP)- or peroxidase (POD)-conjugated anti-mouse Ig antibody.P Bound anti-mouse Ig-AP or anti-mouse Ig-POD is detected by a substrate reactionand visualized by light microscopy.G Table 23: Summary of methods to study cell cycle-associated antigens in individual cells.100101Cell Proliferation and ViabilityMethod/Roche Molecular Biochemicals product23.1Technical tips3.1.13.1.23.1.3Selected frequently asked questions (FAQs) about cell death assaysTechnical tips on the TUNEL methodTechnical tips on the use of Annexin-V-Biotin for lightmicroscope detectionTechnical tips on the use of the Apoptotic DNA Ladder Kit ontissue samplesTechnical tips on the Cell Proliferation ELISA kits3.1.43.1.53.2Special applications of cell death and cellproliferation methods10410410510931091101113.2.13.2.23.2.33.2.4TUNEL assaysMetabolic assaysAnnexin assaysBrdU assays1111131131143.3References3.3.13.3.23.3.3Apoptosis-related parameters – Abbreviations and ReferencesExamples for applications of Boehringer Mannheim productsGeneral references3.4General abbreviations1293.5Ordering Guide1313.6Index134115115120127Chapter 3AppendixTechnical tipsSelected frequently asked questions (FAQs) about cell death assays3.1 Technical tipsNote: For further tips on obtaining thebest results with the TUNEL method,see page 113 of this Appendix.3.1.1 Selected frequently askedquestions (FAQs) about cell deathassaysThe questions below were chosen fromthose received by our Technical Servicesrepresentatives.
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