H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 74
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For example, if an antibody specific to a certain cell-surface molecule is linked to a fluorescent dye, any5.5 • Purification of Cells and Their PartsCell suspensionSheath fluidFilterCondenserFluorescentlightdetector12Scatteredlightdetector231Laser beam−−==−−+Drops withno charge4Drops withlesser chargeFluorescent cellsDrops withgreater chargeNonfluorescent cellSorted chargeddroplets containingfluorescent cellsFluorescent cell dropletsNonfluorescent cell dropletcell bearing this molecule will bind the antibody and willthen be separated from other cells when it fluoresces in theFACS.
Having been sorted from other cells, the selected cellscan be grown in culture.The FACS procedure is commonly used to purify the different types of white blood cells, each of which bears on itssurface one or more distinctive proteins and will thus bindmonoclonal antibodies specific for that protein. Only the T104Green fluorescenceThy1.2 EXPERIMENTAL FIGURE 5-34Fluorescence-activated cell sorter (FACS)separates cells that are labeleddifferentially with a fluorescent reagent.Step 1 : A concentrated suspension oflabeled cells is mixed with a buffer (thesheath fluid) so that the cells pass single-filethrough a laser light beam. Step 2 : Both thefluorescent light emitted and the lightscattered by each cell are measured; frommeasurements of the scattered light, the sizeand shape of the cell can be determined.Step 3 : The suspension is then forcedthrough a nozzle, which forms tiny dropletscontaining at most a single cell.
At the timeof formation, each droplet is given a negativeelectric charge proportional to the amount offluorescence of its cell. Step 4 : Dropletswith no charge and those with differentelectric charges are separated by an electricfield and collected. It takes only millisecondsto sort each droplet, and so as many as 10million cells per hour can pass through themachine.
In this way, cells that have desiredproperties can be separated and then grown.[Adapted from D. R. Parks and L. A. Herzenberg,1982, Meth. Cell Biol. 26:283.]cells of the immune system, for instance, have both CD3 andThy1.2 proteins on their surfaces. The presence of these surface proteins allows T cells to be separated easily from othertypes of blood cells or spleen cells (Figure 5-35). In a variation of the use of monoclonal antibodies for separating cells,small magnetic beads are coated with a monoclonal antibodyspecific for a surface protein such as CD3 or Thy1.2. Onlycells with these proteins will stick to the beads and can be EXPERIMENTAL FIGURE 5-35 T cells bound tofluorescence-tagged antibodies to two cell-surface proteinsare separated from other white blood cells by FACS.
Spleencells from a mouse reacted with a fluorescence-taggedmonoclonal antibody (green) specific for the CD3 cell-surfaceprotein and with a fluorescence-tagged monoclonal antibody (red)specific for a second cell-surface protein, Thy1.2. As cells werepassed through a FACS machine, the intensity of the green andred fluorescence emitted by each cell was recorded. This plot ofthe red fluorescence (vertical axis) versus green fluorescence(horizontal axis) for thousands of cells shows that about half ofthe cells—the T cells—express both CD3 and Thy1.2 proteins ontheir surfaces (upper-right quadrant).
The remaining cells, whichexhibit low fluorescence (lower-left quadrant), express onlybackground levels of these proteins and are other types of whiteblood cells. Note the logarithmic scale on both axes. [Courtesy ofT cells103102101100100179Non-T cells101102CD3Red fluorescence103104Chengcheng Zhang.]180CHAPTER 5 • Biomembranes and Cell Architectureers in which the cells are forced through a very narrow spacebetween the plunger and the vessel wall. As noted earlier,water flows into cells when they are placed in a hypotonicsolution (see Figure 5-18). This osmotic flow causes cells toswell, weakening the plasma membrane and facilitating itsrupture.
Generally, the cell solution is kept at 0 C to bestpreserve enzymes and other constituents after their releasefrom the stabilizing forces of the cell.Disrupting the cell produces a mix of suspended cellularcomponents, the homogenate, from which the desired organelles can be retrieved. Homogenization of the cell and dilution of the cytosol cause the depolymerization of actinmicrofilaments and microtubules, releasing their monomericsubunits, and shear intermediate filaments into short fragments. Thus other procedures, described in Chapters 19 and20, are used to study these important constituents. Because ratliver contains an abundance of a single cell type, this tissue hasbeen used in many classic studies of cell organelles.
However,the same isolation principles apply to virtually all cells and tissues, and modifications of these cell-fractionation techniquescan be used to separate and purify any desired components.recovered from the preparation by adhesion to a small magnet on the side of the test tube.Other uses of flow cytometry include the measurement ofa cell’s DNA and RNA content and the determination of itsgeneral shape and size. The FACS can make simultaneousmeasurements of the size of a cell (from the amount of scattered light) and the amount of DNA that it contains (from theamount of fluorescence emitted from a DNA-binding dye).Disruption of Cells Releases Their Organellesand Other ContentsThe initial step in purifying subcellular structures is to rupture the plasma membrane and the cell wall, if present.
First,the cells are suspended in a solution of appropriate pH andsalt content, usually isotonic sucrose (0.25 M) or a combination of salts similar in composition to those in the cell’sinterior. Many cells can then be broken by stirring the cellsuspension in a high-speed blender or by exposing it toultrahigh-frequency sound (sonication).
Plasma membranescan also be sheared by special pressurized tissue homogenizFilterhomogenateto removeclumps ofunbrokencells,connectivetissue, etc.Centrifuge600g ×10 minFilteredNucleihomogenatePour out:15,000g ×5 minPour out:100,000g ×60 minMitochondria,chloroplasts,lysosomes,andperoxisomes▲ EXPERIMENTAL FIGURE 5-36 Differential centrifugationis a common first step in fractionating a cell homogenate.The homogenate resulting from disrupting cells is usually filteredto remove unbroken cells and then centrifuged at a fairly lowspeed to selectively pellet the nucleus—the largest organelle.The undeposited material (the supernatant) is next centrifuged ata higher speed to sediment the mitochondria, chloroplasts,lysosomes, and peroxisomes.
Subsequent centrifugation in thePour out:300,000g ×2hPlasmamembrane,microsomalfraction(fragments ofendoplasmicreticulum),and largepolyribosomesPour outRibosomalsubunits,smallpolyribosomesSolublepart ofcytoplasm(cytosol)ultracentrifuge at 100,000g for 60 minutes results in depositionof the plasma membrane, fragments of the endoplasmicreticulum, and large polyribosomes. The recovery of ribosomalsubunits, small polyribosomes, and particles such as complexesof enzymes requires additional centrifugation at still higherspeeds. Only the cytosol—the soluble aqueous part of thecytoplasm—remains in the supernatant after centrifugation at300,000g for 2 hours.5.5 • Purification of Cells and Their PartsCentrifugation Can Separate Many Typesof OrganellesIncreasing density ofsucrose (g/cm3)In Chapter 3, we considered the principles of centrifugationand the uses of centrifugation techniques for separating proteins and nucleic acids. Similar approaches are used for separating and purifying the various organelles, which differ inboth size and density and thus undergo sedimentation at different rates.Most cell-fractionation procedures begin with differential centrifugation of a filtered cell homogenate at increasingly higher speeds (Figure 5-36).
After centrifugation ateach speed for an appropriate time, the supernatant ispoured off and centrifuged at higher speed. The pelletedfractions obtained by differential centrifugation generallycontain a mixture of organelles, although nuclei and viralparticles can sometimes be purified completely by this procedure. An impure organelle fraction obtained by differential centrifugation can be further purified by equilibriumdensity-gradient centrifugation, which separates cellularcomponents according to their density. After the fractionis resuspended, it is layered on top of a solution that con-Organellefraction1.091.111.151.191.221.25BeforecentrifugationLysosomes(1.12 g/cm3)Mitochondria(1.18 g/cm3)Peroxisomes(1.23 g/cm3)Aftercentrifugation▲ EXPERIMENTAL FIGURE 5-37 A mixed organellefraction can be further separated by equilibrium densitygradient centrifugation.
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