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Biology 2382B

Cell Bio Review: Techniques: • Cell Culture - the technique used to grow cells or tissues outside the organisms under strictly controlled conditions - trypsin and proteolytic enzymes - break the cell-cell and cell-matrix interactions adn then you can culture those cells Two types of cells that we can culture: - primary cell culture is something extracted from tissue, while a cell line is immortal Embryonic Stem Cells: - egg and sperm fuse together, adn tit divides until it reaches a stage call blastcyst (unique because they contain inner-cell mass cells) - in order to adequately culture embryonic stem cells we have to feed them with nutrients (do this by pro- viding them with the fibroblast feeder) Adult Stem Cells: - finger-like projections call crypts (where our intestinal stem-cell niche is) - mesenchymal cells are signaling the intestinal cells to either divide or differentiate - whether its in vitro or in vivtor, the most important part of harvesting stem cells, are it’s actual niche (it’s microenvironment) iPS Cells: - take any adult somatic cell and by infecting them with the four factors (Oct4, Sox2, Klf4, Myc, we can de- differentiate these cells into desired tissue type - so they’ll come pluripotent cells Self renewal of adult stem cells is maintained by their interaction with: a. tumor cells b. restricted potential stem cells c. progenitor cells d. stem cell niche e. blood cells Tools of the Trade to Visualize Cells: - Atomic Force Microscopy - makes a 3D image, very high resolution, very high magnification - Resolution (D): ability to distinguish between two very closely positioned objects *** smaller D = the better resolution! • Upright and Inverted Microscopes - inverted: objective is below the stage, while in upright the objective is above the stage Light Microscopy: Bright-field Microscope - view live unstained/stained cells Phase-Contrast Microscopy: - examine live unstained cells - phase plate refracts a portion of the light - interference: two wave lengths interfere with each other which introduces contrast into our image - recognize the different plates Differential Interference CONTRAST Microscopy: ** LOOK ON WEBCT Question: What is the purpose of using an inverted microscope in a cell biology lab? a. provides better conditions for viewing stained cells b. allows viewing live adherent cells in cell culture flask c. it is required for flourescence observations d. it increase the resolution e. it increase the magnification of light microscope Fluorescence Microscopy: - dependent on fluorophores (molecules that when exposed to UV light become excited (unstable) and we can visualize this instability as an emission of fluorescent light How are fluorescence images obtained? - know about the dichoric mirror, EMF and EXF - flourophores emit different signals in different regions Monocilonal Antibodies: - use specific antibodies to recognize an individual protein - make these antibodies by taking an antigen on protein of interest and inject it into spleen cells of a live mouse, so they will make antibodies again antigen we injected, after a couple days, extract spleen cells and culture them, they’re a primary cell culture and you want to turn it into a cell line and you do this by fusing spleen cells with myelonal cells (Cancer cells) and plate mixture of HAT plate, only fused cells (hy- bridomas) can live on HAT media, culture the colonies and test them by using a western blot *know why we use primary and secondary antibodies - inject catalase in mouse, it makes antibodies for it, therefore the primary antibody that you just made in the mouse is the primary one, the secondary antibody recognize only primary antibodies and they are conjugated with different molecules (such as fluorophores, etc.) Question: Monoclonal antibodies can be use in combination with? a. microarray analysis b. southern blot analysis c. western blot analysis d. northern blot analysis e. all of the above Which of the following listed blow is/are true regarding conventional florescent microscopy a. excitation filter is responsible for filtering emitted fluorescence b. light source is polarized c. used to visualize live and fixed samples d. flourophores bind directly to epitopes e. c and d Image Acquisition: - confocal vs conventional - confocal you’re able to focus on an individual focal plane - in conventional you can’t see depth - in confocal you can highlight the individual confocal plane so you can determine depth How many of the following directly apply to a fluorescent microscope? Scanning coils; excitation filter; electrons beam; phosphorescent screen; objective lens; dichroic mir- ror a. 1 b. 2 c. 3 d. 4 e. 5 - electron microscopy gives us a very high resolution Types of EM’s - understand the two different types of electron microscopes: TEM AND SEM - TEM you take histological sections of your samples, and with SEM you can visualize a 3D image - scanning coils in SEM (create very unfavourable environment for electrons) - with TEM we take our sample of interest, dehydrate it (can’t have water with lazer) coat it with heavy metals and section and put it on a copper grid which acts as a stencil for electron beam to get an image on our phosphorescence screen - if we conjugate an antibody with a heavy metal and then treat our sample we can visualize this under TEM (this will be more dense then what we use to coat our specimen) Monoclonal antibodies can be used in combination with TEM to determine: a. detect actively transcribed genes b. produce secondary electron c. fractionate cells into organelle d. emit florescence e. identify the sub-cellular localization of proteins Differential Centrifugation: - as we increase the speed, we can collect smaller organelles Equilibrium Density-Gradient Centrifugation: - know how we can insure that we collected our organelles of interest - take a pipetted, suck sucrose out, and microscope it OR by use of different enzymes you can detect the enzyme activity - with each organelle there are specific enzymes that we can test their activity to determine if in fact we’ve isolated the correct organelle How are proteins separated from the organelle? - memorize SDS and Triton X - ionic: denature, non-ionic do not SDS-PAGE: - separate based on molecular wait How to detect a specific protein? - apply specific antibodies to recognize a particular protein of interest - all the band tells us is that there are proteins present with this molecular weight, we don’t know how many different types of proteins present Isoelectric Focusing: - focus our proteins before we run them on a PAGE - IEF - determines a proteins isoelectric points (net charge=0) 2D gel electrophoresis: - size and charge Question: In 2D electrophoresis proteins are first resolved by ___ and then by ___ a. SDS page; affinity chromotgraphy b. SDS page; ion exchange c. IEF; gel filtration d. IEF; SDS PAGE e. SDS- PAGE; wester blot Isolation of Biologically-Active Proteins by Column Liquid Chromatography - gel filtration: size - ion-exchange: charge - affinity: we can conjugate beads with a desired antibody Y2H: - detecting protein-protein interaction - protein is called the bait Take Home Message: 1. Protein-Protein 2. Transfect BAIT + F1, BAIT+F2...BAIT-F# 3. First selectable marker The finals creen using yeast two -hybrid clonign to detect protein:protein interactiosn requires taht the recombinant yeast cells bei incuabated: a. in a medium containing leu and trp b. medium containg his c. lacking leu and trp but containing his d. lacking his e. containing leu trp and his DNA Microarrays: - detect global (the overall) gene expression detect global gene expression (the overall gene expression) comparing differences in gene expression in normal cell line vs. cancerous cell line what genes are being turned on in cancerous cell line that is allowing for this phenotype extract total mRNA, reversely transcribe them to get cDNAs, and in doing so we fluorescently label the normal cDNA one colour, and the cancerous one another colour we also have a chip which has microscopic wells and inside each well contains single stranded DNA, each well contains the sequence of a given gene (it is genomic DNA that is the correct sequence of a giv- en gene that could be expressed in this cell type) add the mixture of fluorescently labeled cDNA from both the normal and cancerous cell lines, and detect whether or not the genes are being expressed complimentary DNA will hybridize to the single stranded DNA fragments
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