Apoptosis and Cell Proliferation (522915), страница 11
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No.TUNEL Label Mix1 767 2913 x 550 µl (30 tests)TUNEL Enzyme1 767 3052 x 50 µl (20 tests)TUNEL POD (Anti-Fluorescein, POD conjugate)1 772 4653.5 ml (70 tests)TUNEL AP (Anti-Fluorescein, AP conjugate)1 772 4573.5 ml (70 tests)TUNEL Dilution Buffer1 966 0062 x 10 mlDAB Substrate, metal enhanced, precipitating (POD substrate)1 718 0961 packNBT/BCIP Stock Solution (AP substrate)1 681 4518 mlFast Red Tablets (AP substrate)1 496 54920 tabletsG Table 6: Single reagents available for the TUNEL technique.30Pack Size1.2.2.2 Assays that measure membranealterationsIn contrast to necrosis, apoptosis occurswithout inflammation.
In the end stages ofapoptosis, apoptotic bodies are engulfed bymacrophages and other phagocytic cells2in vivo. Thus, apoptotic cells are removedfrom the population without spilling theircontents and eliciting an inflammatory response.The exact mechanism by which the apoptotic cell becomes a target for phagocytes isunclear. However, it has been shown that anumber of changes in cell surface (membrane) markers occur during apoptosis, anyone of which may signal “remove now” tothe phagocytes. These membrane changesinclude:P Loss of terminal sialic acid residuesfrom the side chains of cell surface glycoproteins, exposing new sugar residues23, 24.P Emergence of surface glycoproteins thatmay serve as receptors for macrophagesecreted adhesive molecules such asthrombospondin25.In theory, any of these membrane changescould provide an assay for apoptotic cells.In fact, one of them has – the alteration inphospholipid distribution.In normal cells (Figure 22, left diagram),the distribution of phospholipids is asymmetric, with the inner membrane containing anionic phospholipids (such as phosphatidylserine) and the outer membranehaving mostly neutral phospholipids.
Inapoptotic cells (Figure 22, right diagram)however, the amount of phosphatidylserine (PS) on the outer surface of the membrane increases, exposing PS to the surrounding liquid27.Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure membrane alterations1Annexin V, a calcium-dependent phospholipid-binding protein, has a high affinity forPS27. Although it will not bind to normalliving cells, Annexin V will bind to the PSexposed on the surface of apoptotic cells(Figure 23, 24).
Thus, Annexin V hasproved suitable for detecting apoptoticcells28, 29. Roche Molecular Biochemicalssupplies a number of products for the detection of PS translocation by Annexin V.P Loss of asymmetry in cell membranephospholipids, altering both the hydrophobicity and charge of the membranesurface26.G Figure 22: Detection of surface morphology changes during apoptosis. During apoptosis, the distribution ofneutral phospholipids (black symbols) and anionic phospholipids such as phosphatidylserine (red symbols) in the cellmembrane changes. Phosphatidylserine is present in the outer membrane of apoptotic cells, but not of normal cells.An exogenously added molecule specific for phosphatidylserine, such as Annexin-V-FLUOS, will bind to phosphatidylserine on the outer membrane of apoptotic cells, but cannot react with the phosphatidylserine of normal cells.31Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure membrane alterationsAnnexin-V-FLUOSCat.
No. 1 828 681250 testsAnnexin-V-FLUOS Staining KitCat. No. 1 858 777150 testsAnnexin-V-Alexa™ 568Cat. No. 1 985 485250 testsTypeDirect fluorescence staining for flow cytometric or microscopic analysisUseful forDetection of apoptotic cells with membrane alterations (phosphatidylserinetranslocation); differentiation of apoptotic from necrotic cellsSamplesCell lines (adherent or suspensions), freshly isolated cellsMethodSimultaneous staining of cell surface phosphatidylserine [with Annexin-VFLUOS (green dye) or Annexin-V-Alexa™ 568 (red dye)] and necrotic cells(with propidium iodide)TimeApprox. 15 min (after induction of apoptosis)Significance of reagent and kit: AnnexinV is a phospholipid-binding protein with ahigh affinity for phosphatidylserine (PS).Detection of cell-surface PS with annexin Vthus serves as a marker for apoptotic cells.Analysis may be by flow cytometry or byfluorescence microscopy.Test principle: Annexin-V-FLUOS (greendye) and Annexin-V-Alexa™ 568 (red dye)serves as a fluorescent probe for apoptoticcells.
They will not bind normal, intactcells. However, since necrotic cells areleaky enough to give Annexin-V-FLUOSand Annexin-V-Alexa™ 568 access to innermembrane PS, apoptotic cells have to bedifferentiated from necrotic cells. Thus, theassay involves simultaneous staining withboth Annexin-V-FLUOS and the DNAstain propidium iodide or Annexin-VAlexa™ 568 and BOBO™-1 (or propidium iodide). Exclusion of propidium iodideor BOBO™-1, coupled with bindingof Annexin-V-FLUOS or Annexin-VAlexa™ 568, indicates an apoptotic cell(Table 7). The procedure (Flow Chart 7)involves:321Washing suspended cells, then pelletingthe cells.2Resuspending cells in a staining solutioncontaining Annexin-V-FLUOS andpropidium iodide or Annexin-VAlexa™ 568 and BOBO™-1.Note: Cells may also be labeled withother membrane stains, such as a fluorescein-, phycoerythrin- or TRITC-labeledmonoclonal antibody simultaneously.3Analyzing samples in a flow cytometeror under a fluorescence microscope.NormalcellsApoptoticcellsNecroticcellsAnnexin-Vstaining–++Propidiumiodidestaining––+G Table 7: Distinguishing apoptosis using Annexin-V.Treat sample (106 cells) with apoptosis-inducing agent(1–24 h)A3:DATA036\FL1-H\FL1-HeightU937 + Annexinfluos + PJBC3:DATA036\FL2-H\FL2-HeightU937 + Annexinfluos + PJ3:DATA036U937 + Annexinfluos + PJWash treated cells with PBS and centrifuge (200 x g )(5 min, RT)Incubate cells in binding buffer containing Annexin-VFLUOS and propidium iodide or Annexin-VAlexa™ 568 and BOBO™-1.
(10–15 min, RT)Add binding buffer tostained cells3:DATA038\FL1-H\FL1-HeightU937 + Annexinfluos + PJ + CAM3:DATA038\FL2-H\FL2-HeightU937 + Annexinfluos + PJ + CAM3:DATA038U937 + Annexinfluos + PJ + CAM1Analyze by fluorescencemicroscopyAnalyze by flow cytometryG Flow Chart 7: Assay procedure, Annexin-V-FLUOSStaining Kit and Annexin-V-Alexa™ 568.Specificity: Annexin-V-FLUOS and Annexin-V-Alexa™ 568 bind apoptotic cellsand leaky necrotic cells. Propidium iodideand BOBO™-1 are excluded from apoptotic and normal cells, but is taken up by necrotic cells.G Figure 23: Flow cytometric analysis of apoptotic U937 cells stained with Annexin-V-FLUOSand propidium iodide. U937 cells (a leukemic cell line) were cultivated for 4 h with (bottom row) orwithout (top row) 4 µg/ml campothecin.
Cells were then stained with the components of the AnnexinV-FLUOS Staining Kit and analyzed.Panels A (upper and lower), single parameter analysis, Annexin-V-FLUOS only;Panels B, single parameter analysis, propidium iodide (PI) only;Panels C, dual parameter analysis, Annexin-V-FLUOS and propidium iodide. FL1, Annexin-V-FLUOS;FL2, propidium iodide.Result: Flow cytometric analysis clearly differentiates normal (living) cells (R1) with low Annexin andlow PI staining, apoptotic cells (R2) with high Annexin and low PI staining, and necrotic cells (R3) withhigh Annexin and high PI staining.Can be used to assay:P Cell lines (adherent or suspensions)AP Freshly isolated cellsReagent contents:P Annexin-V-FLUOS solution, 50 x concentratedP Annexin-V-Alexa™ 568, 50 x concentratedKit contentsAnnexin-V-FLUOS Staining Kit1.
Annexin-V-FLUOS, 50 x concentrated2. Propidium iodide solution, 50 x concentrated3. Binding buffer for flow cytometry,ready to useCell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure membrane alterationsBTypical results: See Figures 23 and 24.CF Figure 24: Fluorescent microscopic analysis of apoptotic SKW6.4cells stained with Annexin-V-FLUOS,DAPI, and propidium iodide. SKW6.4cells were treated with anti-fas antibody, then stained with a series of fluorescent dyes.Panel A: Cells stained with Annexin-VFLUOS (green)Panel B: Same slide, stained with DAPI(blue)Panel C: Another slide, stained withpropidium iodide (orange) and AnnexinV-FLUOS (green).Result: A few apoptotic cells are visiblein panel A (bright green stain) and canbe differentiated from necrotic cells bythe propidium iodide staining in panel C.(Necrotic cells take up propidium iodideand stain orange/green, while apoptoticcells stain green only).
An additionalstain, DAPI (panel B) shows that theapoptotic cells have characteristiccondensed nuclei .Note: A and B are identical cells,C is different.33Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure membrane alterationsAnnexin-V-BiotinCat. No. 1 828 6901250 testsTypeIndirect fluorescence staining for flow cytometric, fluorescence or lightmicroscopic analysisUseful forDetection of apoptotic cells with membrane alterations (phosphatidylserinetranslocation); differentiation of apoptotic from necrotic cellsSamplesCell lines (adherent and suspensions), freshly isolated cellsMethodSimultaneous staining of cell surface phosphatidylserine (with Annexin-VBiotin) and necrotic cells (with propidium iodide), followed by detection ofbiotin (with streptavidin/avidin conjugate)TimeApprox.
75 min (after induction of apoptosis)Significance of reagent: Annexin V is aphospholipid-binding protein with a highaffinity for phosphatidylserine (PS). During apoptosis, PS translocates to the outersurface of apoptotic cells. Detection of cellsurface PS with annexin V thus serves as amarker for apoptotic cells. Labeling of cellswith the Biotin-conjugate of Annexin-V allows fixation after Annexin-V binding forfurther analysis of additional cellular parameters in combination with detection ofapoptosis (van Engeland, M., RamaekersFCS, Schutte, B & Reutelingsperger, CPM(1996): A Novel Assay to Measure Lossof Plasma Membrane Asymmetry DuringApoptosis of Adherent Cells in Culture.Cytometry 24: 131–139).
For distinguishing apoptosis using Annexin-V, see Table 7,page 32.Test principle: Annexin-V-Biotin serves asa probe for apoptotic cells. It will not bindnormal, intact cells. However, since necrotic cells are leaky enough to give AnnexinV-Biotin access to inner membrane PS,apoptotic cells have to be differentiatedfrom necrotic cells. Thus, the assay involves simultaneous staining with both Annexin-V-Biotin and propidium iodide.
Ex-34clusion of propidium iodide, coupled withbinding of Annexin-V-Biotin, indicates anapoptotic cell. Annexin-V-Biotin is visualized with a streptavidin conjugate. Analysismay be by flow cytometry, by fluorescencemicroscopy, or by light microscopy. Theprocedure (Flow Chart 8) involves:1Washing suspended cells, then pelletingthe cells.2Resuspending cells in a staining solutioncontaining Annexin-V-Biotin and propidium iodide.Note: Cells may also be labeled withother membrane stains, such as a fluorescein-, phycoerythrin- or TRITC-labeled monoclonal antibody simultaneously.3Washing labeled cells.4Incubating cells with a streptavidin (SA)/avidin (A) conjugate (Table 8).5Analyzing samples in a flow cytometer,under a fluorescence microscope, or under a light microscope (depending onthe SA conjugate).F Flow Chart 8: Assay procedure,Annexin-V-Biotin.Treat sample (106 cells) with apoptosis-inducing agent (1–24 h)Wash treated cells with PBS and centrifuge (200 x g ) (5 min, RT)Incubate cells in incubation buffer containing Annexin-V-Biotin and propidium iodide (10–15 min, RT)Centrifuge stained cells (200 x g ) and wash once with incubation buffer (approx.
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