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cell bio summary notes - Test 1.docx

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Department
Biology
Course Code
Biology 2382B
Professor
Robert Cumming

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Lecture 1 What is a cell?  Eukaryotic cells have nuclei, prokaryotic don’t Hypothesis-Driven Experiments  First need to isolate + maintain cells in vitro  Then need to view cells  Then study how proteins drive biological processes Cell Culture  Technique for growing cells/tissues outside organism under strictly controlled conditions  Isolate cells from tissues by breaking cell-cell + cell-matrix interactions o Cells are sticky and stick together tightly, need to loosen them up so that they can be isolated o Use mechanical fragmentation, trypsin (enzyme that goes around the cells and breaks up the proteins on the surface that keep cells stuck together), EDTA (divalent cation chelator that depletes Ca+ in the medium that is needed by cell-adhesion molecules)  Grow isolated cells in media containing necessary nutrients (amino acids, minerals, vitamins, salts, glucose) and serum (insulin - helps take up glucose; and growth factors)  tissue culture media o Grow the cells in tissue culture incubator which has conditions similar to the human body (or wherever the cells came from)  37 degrees, CO2, O2 o The tissue culture media contains pH indicator dye (phenol red) o Leave cells in incubator overnight  If the glass is charged, then cells will stick to the bottom  If you want cells in suspension then you need to keep in motion via magnetic spinner or roller bottles  If just left there then cells will naturally float to the bottom due to gravity  Monolayer = single layer that is one cell thick; confluent = all cells are touching each other to form monolayer; contact inhibition = cells stop growing because they know they’re all touching each other o If you broke the confluent monolayer using trypsin, the cells would grow again until they reached confluency again; but cells can only do this limited number of times before the cells senesce o Immortal cells can keep growing/divide indefinitely; they’re less likely to undergo contact inhibition and will grow on top of each other in mounds  Primary cell culture: cells taken directly from organism, they divide a limited number of times, undergo contact inhibition  Cell line: cells that have been transformed and are thus immortal cells, grow indefinitely, do not undergo contact inhibition o HeLa was the first human cell line (no contact inhibition)  caused cancer Morphology of normal vs. transformed fibroblasts  Normal cells: elongated, aligned, orderly packed, grow in parallel arrays, contact inhibition to stop growing  Transformed: rounded, hair-like extensions, disorganized, grow on top of each other, loss of contact inhibition o Transformed the normal cells with a gene that disrupts normal contact inhibition  This way you can transform primary cells into a cell line Birth, Lineage, and Death of Cells  All cells originate from stem cells; early stem cells come from developing embryo  Stem cell grows then symmetrically divides in two, then can go in 1 of 3 ways: o Grows to become a precursor/late stem cells then that asymmetrically divides to differentiated cells  mature functional cells that participate in some tissue processes; undergo changes to gene expression to define function of the cell o Give birth to a cell lineage (keeps dividing to form more stem cells; but at any point in the lineage the cell could differentiate o Cell death could be programmed to either stem cells or differentiated cells Embryonic stem cells can be maintained in culture and can form differentiated cells  First take blastocyst and remove inner cell mast (group of cells inside)  Place the isolated inner cell mast on a confluent monolayer of fibroblast feeder cells in a petri dish (culture) – fibroblasts provide growth factors and other things that help the cell survive  Only a few of those original cells survive in the culture  Use trypsin to dissociate those cells that survive and put them in a new culture with new monolayer of fibroblasts  These grow into embryonic stem cell cultures  ES cells ARE pluripotent (can give rise to many types of cells) and can be grown indefinitely in the culture under appropriate conditions; we can alter the conditions so that they differentiate  Embryoid bodies are 3D aggregates of pluripotent stem cells, basically what you get after you put the surviving ES cells into a culture o Embryoid bodies have characteristics similar to ES cells in that they can differentiate and keep dividing o Embryoid bodies can be put into a suspension to be used for various purposes o ES cells within embryoid bodies differentiate into 1 of 3 germ lines: endoderm, mesoderm, and ectoderm  comprise all somatic cells Adult Stem Cells  Most tissues contain adult stem cells for replenishing/repairing worn out tissues; with age, the stem cells do not work as well and the tissues do not get repaired as effectively  AS cells are NOT pluripotent – give rise to only a limited number of cell types  Adult stem cells are found in stem-cell niche (cells beside the stem cells called accessory cells, and those help determine whether the stem cell will renew itself or differentiate) o Ex. intestinal stem cells:  There are hills (villi) and valleys (crypt); intestinal stem cells are found in the crypt; they replenish the intestinal epithelium by pushing new stem cells up towards the villi to shed off the old ones Induced Pluripotent Cells  There is a small number of stem cells, but lots of different differentiated cells  Pluripotent stem cells can be obtained from differentiated normal cells; called induced pluripotent stem cells (iPS cells)  Adding Oct4, Sox2, Klf4 genes (typical of ES cells) and c-Myc gene (expressed in cancer cells) will reprogram a differentiated cell into an iPS cell  After transcription and translation of these genes, the resulting proteins will activate genes for self-renewal, pluripotency, and also repress genes that induce specific differentiation pathways  May lead to good replacement of transplants because transplants come from other people (foreign cells), while iPS cells are from own body Origins of Cancer  Normal cells are beneficial because they have function - they come from stem cells; stem cells are unspecialized, self renew, and can generate specialized cells, but they themselves cannot function  Genetic mutations can arise due to environmental factors, resulting in uncontrolled cell division at checkpoints and prevent cell death (apoptosis) o Cancer needs uncontrolled growth + inability to program cell death  Transformation (change in genetic characteristics of a cell) of a stem cell or a normal cell can result in cancer  Cancer cells from stem cells are worse because they still have some properties of stem cells and may differentiate into many TYPES of cancers  Reprogramming normal cells to stem cells could potentially promote transformation to cancer cells  Immortal cells commonly come from cancer cells Lecture 2: Imaging in Cell Biology Light (bright-field)  Light source from below specimen, shines through microscopy condenser lens, which focuses it through specimen  Objective lens (100x magnification) collects the light that passed through specimen, then focuses it to the eyepiece (ocular; 10x mag)  Eyepiece then focuses light onto eye which sees the product of both magnifications  Ability to see cells is dependent on cell structures; high refractive indexes (slowing/bending of light) are observable Phase Contrast  Used to examine live, unstained cells microscopy  Light source from below specimen, shines light through a condenser lens, which focuses the light through the specimen  Anywhere the light passes through the specimen will slow the wavelength by ¼ , the rest of the light passes through unimpeded along the edge of the cone of light  Another objective focuses the light (impeded + unimpeded) through a phase plate which has a coating that slows the wavelength down another ¼ , light on the edge goes through unimpeded again  The inner rays of light have been slowed down by ½ now, while the outer rays have not been slowed; gives contrast between slowed (darker) and not slowed (lighter) parts  The two phases of light recombine at the eye due to interference, and we can see contrast  Certain parts of the cell (eg. nucleus) refract (slow) light more than other parts  If the wavelength was slowed more, then when recombined with normal wavelength there is more interference = shallower peaks/valleys in recombined wavelength = dim light  More contrast = better ability resolve cells  Gives halo effect so edges of cell are not as clear, but gives more contrast than DIC microscopy Differential  Used to examine live, unstained cells Interference  Uses polarized light; built in polarizers in microscopes Contrast microscopy can select certain angles of light to let through (Normarski  Recombine (interference) polarized light to get microscopy) contrast in overall image; small differences in refractive index + thickness within cell are converted to contrast  Good for showing 3D form of cell, outlines of large organelles, edge of cell, shadows, topography Fluorescence  Uses certain chemicals (fluorophores) that absorb UV microscopy light and emit different colours  Fluorescent proteins/dyes bind to certain structures in cells and can be imaged  Light (300-700nm, very bright + strong) comes from above specimen  Light passes through filter that allows one wavelength of light through; wavelength hits mirror to reach specimen which excites electrons then fall and emit different wavelength of light  Emitted light gets reflected off specimen to projection lens which focuses the emitted light to our eyes Immunofluorescence  Make antibodies specific to proteins that you want to microscopy image  Treat glass that cells stick (die), then wash the cells with the antibody solution; cells are frozen in time so that antibodies can hybridize to YFP  The antibody is not fluorescent so cannot see yet, must make another antibody that contains a fluorophore to recognize the first antibody  Thus can use certain wavelength to excite electrons and image where the anitbodies/proteins are in the cell  LIMITATION: cells are fixed/cannot move/dead Dual Label  Use appropriate microscope filter (wavelength) to excite Fluorescence one fluorophore at a time, then image each separately microscopy and overlay the images  LIMITATION: cells are fixed/cannot move/dead Confocal microscopy  Reduces fuzziness and depth issues found in fluorescence microscopy, good for 3D imaging  Laser ABOVE THE SPECIMEN goes through pinhole to a mirror that reflects the laser point to the cell  Tiny laser point excites small pin points of the cell at a time; only parts of the cell that are excited from that pin point will be passed through another pinhole to detector  After scanning the entire cell point by point, the computer creates composite image of the scanned cell  Gives slightly higher resolution  Laser illuminates only what is on the focal plane (thin optical selection of the cell; sort of like the cross section), thus blocking the influence of out-of-focus light above and below the plane caused by fluorescence of other proteins above and below Deconvolution  Removes fluorescence from out-of-focus parts microscopy (fuzziness), and takes images at more than one focal plane (Z-Stack)  Compares the fluorescence intensities at each level of the Z-stack to subtract the blurriness and focus in on the fluorescence of just that Z-stack  ** deconvolution illuminates Z-Stacks vs. confocal illuminates only the focal plane Electron microscopy  higher resolution and magnification than fluorescence microscopy; but EM requires cells to be dead, fixed, sectioned, metal-coated  NOT USING LIGHT; uses electrons from a wire filament  Wire is heated and electrons accelerated towards positive anode; beam of electrons then focused through MAGNETIC condenser to specimen  Specimen cell structures stained with heavy metals, so when electron beam shined through specimen, the heavy metals don’t allow them to pass  dark spots  Must occur in a vaccum so that electrons do not fly around and get absorbed atoms in the air  Transmission  Condenser lens focuses electron beam THROUGH electron microscope specimen and the electrons that do pass through the (TEM) specimen are focused onto a detector by objective and projector lenses  Shows internal structures  Very small resolution (D): no N because light replaced by electrons in vacuum, no sin alpha because ~0 electron scatter  Still want to keep D small; theoretical D = 0.005nm; effective D = 0.1nm   Scanning electron  Condenser lens focuses electron beam through scanning microscope coils and objective lens to specimen; then the scattered electrons that were reflected by metal-coated specimen are detected to produce a 3D image  Shows external structures Immunoelectron  Instead of using heavy metals, you can use antibodies to microscopy hybridize with the specific proteins in the cell  Heavy metal is then linked to the antibody via some protein A  Then put through electron microscopy Resolution of Microscopes  Resolution: the ability to distinguish between two very closely positioned objects as separate entities  SMALLER RESOLUTION IS BETTER  Detail seen is dependent on resolution of microscope; want to optimize resolution to see finer detail  Naked eye resolution = 2cm; conventional microscope resolution = 0.2μM  Resolution = o D = distance resolved between 2 points  small is good o = Wavelength of light  small is good (use smaller wavelength of light) o = Numerical aperture  big is good o = Refractive index of medium between specimen and objective lens  big is good (use medium with higher refractive index – eg. use water or oil instead of air) o = ½ angle of light entering objective  big is good (bring objective lens closer to specimen = bigger angle)  Limit of resolution is 0.2microns = 200nm  Microscopy uses wavelengths of light in the visible region (400-700nm) Fluorophores  Chemical that can be excited with certain wavelengths of light, causes electrons to jump to excited state (unstable); electron falls back down to ground state and emits a light of DIFFERENT wavelength o Excitation wavelength and emission wavelength o Difference between optimums is called Stokes shift o Optimum is the max wavelength of absorption  Many different colours of fluorophore dyes/proteins can be used to stain different cell structures Monoclonal Antibodies  Antibodies = specific proteins that bind to antigens as a signal to kill the foreign molecule  part of innate immune system  Monoclonal antibodies made by injecting synthetic protein of interest into mouse  Mouse senses YFP as foreign invader and starts producing antibodies in spleen that recognize YFP (antigen)  Remove spleen cells (Bcells stored in nodules)  primary cells, divide limited # of times; want these cells to be immortal though so you can keep producing antibodies, therefore make hybrid cell line  Hybrid cell line made by fusing mutant myeloma cancer cell with spleen cells on HAT medium (toxic for myeloma cells which have mutation of specific gene)  After incubating both cells on the HAT medium all that will be left is hybrids because unfused myeloma cells will die, and spleen cells that do not fuse will die off eventually because they are not immortal  Can then culture the hybrid cells which have characteristics of both cancer cells (immortal) and spleen cells (produce antibody) Fluorescent imaging in LIVE CELLS: Green Fluorescent Protein (GFP)  GFP is protein in jellyfish that contains certain amino acid sequence (chromophore) that fluoresces when blasted with blue (UV?) light  Protein has can-like structure with light in the middle  Using recombinant DNA technology, can fuse GFP gene to YFG (your favourite gene)in an expression plasmid, then transform cells with it  After transcription and translation, produces GFP fused to YFP, and when this fused protein is excited with blue wavelength of light, it emits green wavelength of light  Allows us to see where proteins are active in LIVE cells o Can also mutate GFP to show different colours and fuse it to different proteins, similar to dual label fluorescence microscopy Fluorescence Resonance Energy Transfer (FRET) to Measure Protein INTERACTIONS in LIVE cells  Fuse one protein with cyan fluorescence protein (CFP); fuse another protein with yellow fluorescence protein (YFP)  Using 440nm will excite CFP but not so much YFP; therefore if there is no protein interaction between the two proteins of interest, then only CFP will be excited and emit 480nm light  If there is protein interaction, then the 440nm will excite CFP, which will then produce a wavelength of light (480nm) that excites YFP, which then fluoresces yellow (535nm)  Therefore during live imaging, you can see when the two proteins interact with each other whenever there is yellow fluorescence Light microscopy vs. Electron microscopy  EM uses electromagnietic lenses to focus high-velocity electron beam  Light microscopy uses optical lenses to focus visible light Sample preparation for TEM  Tissue/cells chemically fixed + dehydrated, then embedded in plastic where thin sections of the cell are made  Stain the thin slice of cell with heavy metal  Place preparation in vacuum and view through electron microscope How are Electron Micrographs Formed?  When electrons hit part of the specimen that is stained with heavy metal, it gets deflected  But electrons that do go through the cell are focused by lenses onto phosphorescent screen and the crystals in the screen are excited by the electrons when they hit, give off energy (visible light)  Areas that take up less stain appear lighter  Black and white images are made of shadows where the electrons didn’t penetrate (were reflected) Lecture 3: Isolation and Analysis of Cell Organelles and Molecules Labeling Live Cells with Fluorescent Antibodies or Stains  Usually need to kill cells to view proteins inside the cell; but don’t need to kill cells to view proteins OUTSIDE of cell  Link antibodies specific to cell surface proteins with fluorophores; can also use secondary fluorophore-linked antibodies to bind to protein specific antibodies  Can use membrane permeable dyes to label intracellular structures (Hoechst stain binds DNA in nucleus)  Cells with bound antibodies/stained with dyes can be sorted + counted Fluorescent Activated Cell Sorting (FACS)  Capillary system allows cells to pass through laser beam in single file  Light emitted (fluorescent) and light scattered measured by detectors  As cells drop down individually, metal plates charge cells according to the fluorescent colour that was detected  Charged cells are then sorted (by an electric field) based on their degree of electric charge and collected  Fluorescence, charge, capture based on charge Quantification of Cells Expressing Two Different Cell Surface Markers by FACS  If looking for certain types of cells, label them with certain colour fluorescence, then sort them with FACS  Then can quantify (determine how many cells) have these properties Cell Cycle Analysis by FACS  Hoechst stain goes into cell + binds with DNA; can quantify the degree of blue fluorescence from the stain that is in the cell  Can use intensity of staining to track stages of division (twice the intensity indicates that DNA has replicated - S phase)  Quantifying the number of cells with different staining intensities can give the proportion of cells in G1, S, or G2 How to Study Organelles & Their Proteins  Membranes lead to compartmentalization; all eukaryotes have membrane- bound organelles  Can isolate organelles based on their different density and sizes  Nucleus, mitochondria, and chloroplasts have double membranes How to Isolate Cell Organelles  Need to break apart cell membrane in order to see what is inside: o Homogenizer – tube/pestle pushed up and down, tight space between rod and walls of tube creates strong force to break cell membrane o Sonication – high frequencies break the cell membrane o Pressure – force cells through narrow valve (slightly smaller than size of cell with membrane) o Non-ionic detergents – disrupt membrane (ex. Triton X-100) o Hypotonic solution – creates osmotic difference, water flows inside cell causing it to swell and burst  Centrifuge the Cell Homogenate to sort the stuff inside the cells; differential, equilibrium density-gradient o Rotor spins very fast and uses gravitational forces to force the heavier stuff to the bottom of the tube o Must balance rotor and keep it in vacuum and fridge conditions because it heats up Differential Centrifugation  First filter broken cell membranes and connective tissues out to produce the filter homogenate  Centrifuge homogenate at different speeds and times so that organelles differing in size + mass travel to bottom of tube and pellet there  Can then take the supernatant and centrifuge that at higher speed to get smaller organelles pelleted; increasing centrifugal force (gravity) to isolate organelles based on mass Equilibrium Density – Gradient Centrifugation  Gradient of sucrose densities in tube (low to high), put organelle fraction on top of sucrose, then centrifuge at high speed/several ho
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