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BIOL 4610 1st Edition Lecture 7 Outline of Current Lecture I. Growing Cells in CultureII. Light MicroscopyCurrent LectureChapter 99.1. Growing cells in CultureNutrient requirements – glucose, amino acids, vitamins, salts,1. EUKARYOTIC CELLS NEED GROWTH FACTORSProteins that are used to signal a cell to divideOften come from serum2. Sterility- inside a cabinet, keep out bacteria so divide slower. Also must be at appropriate temperatureSolid supports – most eukaryotic wont grow in Suspension.Petri dish but put a coating like anExtracellular matrix.Polarity or sidednessPrimary cultures – arise from tissueThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.• Finite lifespan- can only replicate so many times Immortal cell lines – grow forever in culture1. In rodents- this can happen spontaneouslyMost die off but a small proportion of them will continue to grow. So we can get them to stay in culture and use them for the next experiment.2. Chemical treatment (mutant DNA)3. Tumor cells, use cancer biopsy and get them to grow forever Separate cells using a flow cytometer or fluorescence activated cell sorter (FACS) –If want a pure culture, we need a way of separating out the cells we don’t want.With the antibody, the Y shaped region recognizes a particular protein that is on a virus. This protein is on the cell of interest (skin cell), but not on another cell type (immune cell). So we can use fluorescently labeled antibodies to recognize the skin cell and stick to them and go thru a straw. Straw only allows 1 cell to pass at a time. Light bulb detects what’s fluorescently labeled by antibody and separates it from the rest. 9.2. Light microscopyNormal bright-field light microscope: • Has a light bulb and uses different lenses to condense light and magnify objectResolution:• See objects down to 0.2 microns• If have 2 objects closer together than 0.2 microns, won’t be able to distinguish them andwill look like 1 object.Phase-contrast microscope:Uses difference in thickness of cellsRefractive index: • light moves more slowly in thicker areas• Ex. If have giant nucleus, light will bend in areas that are thicker• “light moving out of phase”• Areas look darker if out of phase or in thicker areasGood for: • Visualizing single layer cells in culture• Thin tissue slicesNot good for:• Thick tissues, everything will be bentDifferential interference contrast (DIC) microscopes:• Based upon the thickness of cells• Refractive indexFixing and staining cells• Cells have few macromolecules that absorb light well• Other forms of light microscope are not good for thick samples (better for single layers)• Use different stains so it’s easier to look at• “Fix” cells with Formaldehyde- cross links DNA – proteins so macromolecules aren’t degraded• Thick samples: embed in paraffin and then slice into thin sectionsHematoxylin and eosin (2 diff dyes)  Hema. Is basic- (proteins) used for nuclei and stains blue or purple Eosin binds to negatively charge/acidic. Labels the cytoplasm with pink/red


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