Lodish Berk Kaiser Krieger Scott Bretscher Ploegh Matsudaira

Lodish • Berk • Kaiser • Krieger • Scott • Bretscher • Ploegh • Matsudaira MOLECULAR CELL BIOLOGY SIXTH EDITION CHAPTER 9 Visualizing, Fractionating, and Culturing Cells ©Copyright 2008 W. H. ©Freeman andand Company 2008 W. H. Freeman Company

Fluorescence Alpha tubulin (green) Actin (red) DNA (blue) Golgi (yellow) Mitochondria (purple)

Cell Isolation, Culture and Differentiation

Proteases • trypsin Divalent cation chelator • EDTA Senescence • 50 times Cell Strain: line of cell that have a limited life span Cell line: line of cells that are immortal

Media and Controlled Factors: • temperature • atmosphere (pressure) • humidity • p. H • Ionic Strength • Nutrients • essential amino acids • vitamins, salts, fatty acids, glucose, serum • Antibiotics

Flow Cytometry

Hybridomas to produce monoclonal antibodies to specific proteins • Myeloma cell: Immortal • Mouse spleen cell: mortal • Viral glycoproteins/polyethylene glycol • Grown in media that does not allow unfused cells to grow (mutations in metabolic pathways). Fused cells have ability to grow due to sharing of genetic material that was deleted from other cells.

Left: Bright Field Middle: Differential interference contrast Right: Phase Contrast

The table below compares the different types of microscope: http: //universe-review. ca/R 11 -13 -microscopes. htm Characteristic Compound Microscope Transmission E. Microscope Scanning E. Microscope Resolution (Average) 500 nm 10 nm 2 nm Resolution (Special) 100 nm 0. 5 nm 0. 2 nm up to 1, 500 X up to 5, 000 X ~ 100, 000 X poor moderate high living or non-living Preparation Technique usually simple skilled easy Preparation Thickness rather thick very thin variable Specimen Mounting glass slides thin films on copper grids aluminum stubs large enough limited large visible light electrons air vacuum glass 1 electrostatic + a few em. lenses mechanical current in the objective lens coil Magnification Adjustments changing objectives current in the projector lens coil Specimen Contrast by light absorption by electron scattering Magnifying Power Depth of Field Type of Objects Field of View Source of Radiation Medium Nature of Lenses Focusing

Compound microscopes are light illuminated. The image seen with this type of microscope is two dimensional. Description This microscope is the most commonly used. You can view individual cells, even living ones. It has high magnification. However, it has a low resolution. Costs Source of Radiation for Image Formation Medium Specimen mounting Nature of Lenses Focusing Magnification Adjustments Major Means of Providing specimen Contrast Dissection or Stereoscope Confocal Microscope Scanning Electron Microscope (SEM) Transmission Electron Microscope (TEM) TEM is electron illuminated. This A dissection microscope is light SEM use electron illumination. The image This microscope uses a laser light. This gives a 2 -D view. illuminated. The image that appears is is seen in 3 -D. It has high magnification light is used because of the wavelength. Thin slices of three dimensional. It is used for and high resolution. The specimen is Laser light scan across the specimen with specimen are dissection to get a better look at the coated in gold and the electrons bounce the aid of scanning mirrors. Then image is obtained. The larger specimen. You cannot see off to give you and exterior view of then placed on a digital computer screen electron beams pass individual cells because it has a low specimen. The pictures are in black and for analyzing. through this. It has magnification. white. high magnification and high resolution. $150 - $10, 000 $100 -$1500 $20, 000 -100, 000 more than $50, 000 visible light laser light electrons air air vacuum glass slides with dyed samples Mounted on aluminum stubs and are coated in gold Thin films of collodion or other supporting material on copper grids one electrostatic lens and a few electromagnetic lenses glass slides none glass lenses with dichromatic mirrors one electrostatic lens with a few electromagnetic lenses mechanical digital computer motorized focusing mechanism electrical Electrical i. e. current of the objective lens coil is changed. changing objectives usually 1 objective digitally enhanced electrical Electrical i. e. changing current of the projector lens coil Light Absorption light scattering or light reflection laser light with dicromatic mirror concentrated at pinhole electron scattering Electron scattering

Fura-2 Calcium sensitive flourochrome Green: High Ca Blue: Low Ca Why are Calcium concentration moving?

Lysosomes and Mitochondria Green: Mitochondria stained with Mitotracker Green Red: Lysosomes stained with Lyso. Tracker Red

Immunofluorescence Evans Blue Stain (non-specific red) Yellow green fluorescing antibody for GLUT 2 transporters

Deconvolution Fluorescence DNA: Blue Microtubules: Green Actin microfilaments: Red

Plasma Membrane • A microscopic membrane of lipids and proteins that forms the external boundary of the cytoplasm of a cell or encloses a vacuole, and that regulates the passage of molecules in and out of the cytoplasm Mitochondria • An organelle found in large numbers in most cells, in which the biochemical processes of respiration and energy production occur. It has a double membrane, the inner layer being folded inward to form layers (cristae) Lysosomes • An organelle in the cytoplasm of eukaryotic cells containing degradative enzymes enclosed in a membrane Nuclear envelope • Double membrane forming the surface boundary of a eukaryotic nucleus; consists of outer and inner membranes perforated by nuclear pores. Nucleolus • Subcompartment within the nucleus that is involved primarily in making ribosome components. SER • synthesize lipids, steroids and morphine, metabolize carbohydrates and steroids (but not lipids), and regulate calcium concentration, drug metabolism, and attachment of receptors on cell membrane proteins RER • The surface of the rough endoplasmic reticulum (RER) is studded with protein-manufacturing ribosome's giving it a "rough" appearance Golgi Complex • An organelle in eukaryotic cells consisting of stacks of flat membranous sacs that modify, store, and route products of the endoplasmic reticulum.

Secretory Vesicles • A membrane-bound organelle in which molecules destined for export are stored prior to their release, or exocytosis Peroxisomes • A small organelle that is present in the cytoplasm of many cells and that contains the reducing enzyme catalase and usually some oxidases Cytoskeletal Fibers • The cytoskeleton is unique to eukaryotic cells. It is a dynamic three-dimensional structure that fills the cytoplasm. This structure acts as both muscle and skeleton, for movement and stability. The long fibers of the cytoskeleton are polymers of subunits. The primary types of fibers comprising the cytoskeleton are microfilaments, microtubules, and intermediate filaments. Microvilli • microscopic cellular membrane protrusions that increase the surface area of cells, and are involved in a wide variety of functions, including absorption, secretion, cellular adhesion, and mechanotransduction. Cell Wall • usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to act as a pressure vessel, preventing over-expansion when water enters the cell. They are found in plants, bacteria, fungi, algae, and some archaea. Animals and protozoa do not have cell walls. Vacuole • membrane-bound organelle which is present in all plant and fungal cells and some protist, animal and bacterial cells. Vacuoles are essentially enclosed compartments which are filled with water containing inorganic and organic molecules including enzymes in solution, though in certain cases they may contain solids which have been engulfed Chloroplast • found in plant cells and other eukaryotic organisms that conduct photosynthesis. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis.

Electron Micrographs Cultured mammalian cells treated with gold particles (black areas) EE: Early Endosome LE: Late Endosome AV: autophagosomes Rat Liver P: Peroxisomes M: Secondary Lysosome containing mitochondria fragments

Type of Cells/Organ? Profoundness of Glycogen in this cell/location? Random act of placement?

Cell Culture http: //www. invitrogen. com/site/us/en/home/References/gibco-cell-culture-basics. html