Chapter 9 Culturing Cells Growing Cells in Culture 1 Nutrient Requirements glucose amino acids vitamins salts growth factors eukaryotic cells 2 Solid Supports most eukaryotic cells will not grow in a suspension so must have something to adhere to Like adding an extra cellular matrix in a petri dish for cells to grow on If cell has sidedness polarity need to maintain that with specialized supports o Ex Intestinal cells one side interacts with blood other side interacts w nutrients Sterile Technique important when culturing cells Done in a hood to keep manipulations sterile Bacteria cells divide more rapidly so would overcome your cells if end up in sample Primary Cultures those that arise from tissues Designed to have a finite lifespan and only replicate a specific amount of times Usually can keep them in culture for 2 3 weeks Immortal Cell Lines cells that are able to grow in culture forever Can be found sometimes in rodents where immortal cells occur spontaneously Can take primary cells and manipulate DNA with chemical treatments so become immortal Can take biopsies from tumor cells Cells separated by a flow cytometer or florescence activated cell sorter FACS Want a pure culture of just the cells you are interested in Can use antibodies to mark the cells that we want Certain proteins are made in certain cells and can use this to separate cells Antibody is fluorescently labeled and will only bind to the cell of interest marking it Pass cells through very thin tube where only one cell is allowed to pass through at a time o As cell passing through a light detects the antibody and sorts the cells based on if they are fluorescing or not Light Microscopy Normal Light Microscope has light source and uses different lenses to condense light magnify object Does not allow you to see very much inside of the cell Resolution 0 2 microns Phase Contrast Microscope able to see differences in thickness Uses refractive index light moves more slowly through thicker areas bends and light passes right through thin areas Different shades of gray indicate the level of thickness Good for visualizing cells in cultures single layers thin tissue slices Not good for thick tissues or thick slices Differential Interference Contrast DIC Microscope based on cell thickness refractive index Gives more information about the surface characteristics Has a relief map like appearance o Thick areas appear higher and thinner areas appear lower Fixing and Staining Cells to better see the contents of a cell Fixing cross link DNA protein so macromolecules don t degrade often using formaldehyde Staining cells highlights specific organelles or structures that make it easier to see under a microscope Hematoxylin dye that stains basic macromolecules mostly to visualize the nucleus Stains blue purple Eosin dye that stains the most acidic molecules in the cell Labels cytoplasm pink red Florescence Microscopy molecule absorbs light at one wavelength and emits light at a different wavelength Can use it to filter out the wavelengths you aren t interested in 1 Dyes to Detect Intracellular Ion Concentrations using dyes to detect signaling Calcium used in signaling and can be measured with a dye Fura dyes used have ester linkage can passively diffuse into cells o Once inside cells esterases cleave the bond and compound can no longer get out Calcium levels higher outside of cell stimulate cell to let Ca inside and looking for a color change to occur Fura will bind to Ca molecules and emits a different wavelength yellow Immunofluorescence relying on antibodies to detect specific proteins in a cell Antibodies are attracted to specific proteins and because they are fluorescent can identify 2 where those proteins are in the cell under the microscope 3 Recombinant Green Fluorescent Protein Gene GFP recombinant protein Insert gene of interest X into plasmid and also include GFP Every time protein X is made it will then be labeled with GFP 4 Forster Resonance Energy Transfer FRET able to test if proteins interact with each other Make 2 fusion proteins and if the 2 proteins interact can use the emitted wavelength of the first to excite the second protein and if the second protein emits a different wavelength it means that the proteins interacted Electron Microscopy high resolution imaging Transmission Electron Microscopy TEM resolution 0 1 nanometers Good for visualizing very specific structures within a cell Drawback don t get the big picture of the cell only see a small portion of it Beam of electrons is used along with an electromagnetic lens to focus the beam magnify MUST stain cells before using usually with a heavy metal lead gold uranium osmium tetraoxide Cannot be done with living cells Scanning Electron Microscopy SEM specimen moved to the bottom of the electron beam instead of the middle Get a 3D relief appearance with great detail can visualize surfaces Organelles Endosomes membrane bound compartment that originates from plasma membrane Brings proteins macromolecules into the cell via endocytosis Sorts what the cell wants to keep and what it doesn t want to keep Lysosomes only in animal cells Where degradation of macromolecules occurs via enzymes Have a reduced pH 4 5 cytoplasm pH 7 Smooth Endoplasmic Reticulum large network of membranes that are folded into a cisternae Synthesizes lipids vitamins detoxifies enzymes Rough Endoplasmic Reticulum ribosomes attached to it Where protein synthesis occurs Specifically makes proteins designed to go to the plasma membrane those that are secreted from the cell or those that go to specific organelles Proteins leave the rough ER and go to the Golgi Golgi Complex arrive from the ER Helps fold proteins adds sugars disulfide bonds Leaves Golgi through vesicles to protein s final destination