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34 Cards in this Set
- Front
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Light microscopy |
Exploring cell structure and visualizing proteins within cells |
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Electron microscopy (EM) |
High resolution imaging that allows for ultrastructure of |
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Plasma membrane |
Controls movement of molecules in and out of the cell and functions in cell-cell signaling and cell adhesion |
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Mitochondria |
Surrounded by a double membrane, generate ATP by oxidation of glucose and fatty acids Simple: Site of ATP synthesis |
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Lysosomes |
In animal cells only Degrade material internalized by the cell and worn out cellular membranes and organelles Simple: Site of protein degradation |
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Nuclear envelope |
A double membrane enclosing contents of the nucleus |
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Nucleolus |
A nuclear subcompartment where rRNA is synthesized |
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Chromatin |
In nucleus, composed of DNA and proteins |
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Nucleus |
Filled with chromatin and site of mRNA and tRNA synthesis Simple: Site of DNA replicaiton |
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Smooth ER |
Synthesizes lipids and detoxifies certain hydrophobic compounds |
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Rough ER |
Functions in the synthesis, processing, and sorting of secreted proteins, lysosomal proteins, and certain membrane proteins |
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Golgi complex |
Processes and sorts secreted proteins, lysosomal proteins, and membrane proteins synthesized on the rough ER |
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Peroxisomes |
Detoxify various molecules and also break down fatty acids to produce acetyl groups for biosynthesis |
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What should cell media be supplemented with to mimic a cell's natural environment? |
amino acids and growth factors |
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Adherent cells How do they bind? |
Cells that need to adhere to a solid surface to grow Most animal cells Cell surface proteins called cell adhesion molecules that cells use to bind to adjacent cells and to the ECM (extracellular matrix) |
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Suspension cells |
Grow in suspension Blood cells and some tumor cells |
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Primary cells |
Isolated directly from tissue Has finite life span |
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Transformed cells |
Cells derived from tumors Can grow indefinitely in culture |
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Cell line |
A culture of cells with an indefinite life span that is considered immortal |
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Stable cell line |
A cell line modified in the lab to express a specific gene (GFP tag; selection on antibiotics such as Neomyciin) |
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HeLa (Henrietta Lacks) cells |
The first human cell line established Originally obtained in 1952 from a malignant tumor of the uterine cervix |
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Steps to culture cells? |
1. Harvest cells (isolate using appropriate enzymes) 2. Add cells to growth media in a culture dish 3. Incubate at 37 degrees C 4. Subculture cells (every 3-5 days) to obtain a pure culture Cells are then ready for experimental procedures |
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What does flow cytometry do? How is it performed? |
Separates different cell types from a mixture based on their specific surface molecule composition Cell mixture is incubated with a fluorescent dye linked to an antibody for a specific cell surface molecule and analyzed in a flow cytometer |
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FACS (fluorescent activated cell sorter) What kind of cell is it usually used to purify? |
Can analyze the cells, select, and sort them in a separate culture dish after they've been fluorescently labeled White cells --> Can measure the size of a cell and amount of DNA that it contains |
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Steps of FACS? |
1. Labeled cells are suspended and then mixed with a buffer so the cells pass single file through a laser beam 2. Fluorescence and light scattered are measured to determine size and shape of each cell 3. Suspension comes out a nozzle that lets through a one cell droplet at a time --> Each cell is given a negative electric charge corresponding to its fluorescence 4. Droplets pass through an electric field; those with no charge are discarded and those with different electric charges are collected |
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MDCK? |
Can be grown in specialized containers to form a polarized epithelium, which is useful for studying epithelial cells |
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Light microscopy |
Used to determine cell structure and visualize proteins Can visualize unstained living cells and be used in time lapse microscopy to generate a movie |
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What 3 things usually need to be done to image subcellular details? |
Cell/tissue needs to be: 1. Fixed - Use a fixative such as formaldehyde/gluteraldehyde to cross link amino groups on adjacent molecule so the cells are stable for next procedures 2. Permeabilized - Poke holes in cell membrane using non ionic detergent so plasma membrane is permeable to reagents (dyes and antibodies) 3. Stained - fluorescent dyes attach on specific antibodies |
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Define fluorescence When is a chemical fluorescent? |
The molecular absorption of a photon causes the emission of a longer wavelength (could be primary or secondary (treated with chemical capable of fluorescing))
Absorbs light at one wavelength (excitation wavelength) and emits light (fluorescence) at a specific and longer wavelength |
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Immunofluorescence microscopy |
Detects specific proteins in fixed cells |
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Direct immunofluorescence Indirect immunofluorescence |
Antibody is covalently linked to a fluorochrome The specific antibody is detected indirectly --> use secondary Ab that is conjugated with a dye to attach to the other (primary) Ab |
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How was GFP first studied and used? How is GFP used? |
Was purified from the aequorea victoria jelly fish Using recombinant DNA technologies, any protein can be tagged with GFP to obserive its localization and trafficking in the cell |
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Confocal microscopy |
Enhances visualizatoin of fluorescent objects using optical methods to exclude light from other planes so that a specific focal plane can be focused on Fluorescent light emitted by samples comes from not just the plane of focus but also from molecules above and below it |
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FRAP (fluorescence recovery after photo bleaching) |
Used to determine the dynamics of a molecule by bleaching a certain region of a fluorescently labeled area Quantitate recovery of the fluorescence to determine the dynamic property of a protein |
