MOLECULAR CELL BIOLOGY SIXTH EDITION

MOLECULAR CELL BIOLOGY SIXTH EDITION

Copyright 2008 © W. H. Freeman and Company

CHAPTER 9Visualizing, Fractionating,

and Culturing Cells

CHAPTER 9Visualizing, Fractionating,

and Culturing Cells

Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira

© 2008 W. H. Freeman and 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 spanCell line: line of cells that are immortal

Media and Control

Controlled Factors: •temperature•atmosphere (pressure)•humidity•pH•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

Characteristic Compound MicroscopeTransmission E.

MicroscopeScanning E. Microscope

Resolution (Average) 500 nm 10 nm 2 nm

Resolution (Special) 100 nm 0.5 nm 0.2 nm

Magnifying Power up to 1,500X up to 5,000,000X ~ 100,000X

Depth of Field poor moderate high

Type of Objects living or non-living non-living 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

Field of View large enough limited large

Source of Radiation visible light electrons electrons

Medium air vacuum vacuum

Nature of Lenses glass1 electrostatic + a few em.

lenses1 electrostatic + a few em.

lenses

Focusing mechanicalcurrent in the objective lens

coilcurrent in the objective lens

coil

Magnification Adjustments

changing objectivescurrent in the projector lens

coilcurrent in the projector lens

coil

Specimen Contrast by light absorption by electron scattering by electron scattering

The table below compares the different types of microscope:http://universe-review.ca/R11-13-microscopes.htm

Compound Dissection or Stereoscope Confocal MicroscopeScanning Electron Microscope

(SEM)

Transmission Electron

Microscope (TEM)

Description

Compound microscopes are light illuminated. The image seen with

this type of microscope is two dimensional. 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.

A dissection microscope is light illuminated. The image that

appears is three dimensional. It is used for dissection to get a better look at the larger specimen. You

cannot see individual cells because it has a low

magnification.

This microscope uses a laser light. This light is used because of the wavelength. Laser light scan across the specimen with the aid of scanning mirrors. Then image

is then placed on a digital computer screen for analyzing.

SEM use electron illumination. The image is seen in 3-D. It has

high magnification and high resolution. The specimen is

coated in gold and the electrons bounce off to give you and

exterior view of the specimen. The pictures are in black and

white.

TEM is electron illuminated. This

gives a 2-D view. Thin slices of specimen are obtained. The

electron beams pass through

this. It has high magnification

and high resolution.

Costs $150 - $10,000 $100-$1500 $20,000-100,000 more than $50.000 more than $50,000

Source of Radiation for

Image Formationvisible light visible light laser light electrons electrons

Medium air air air vacuum vacuum

Specimen mounting

glass slides none glass slides with dyed samplesMounted on aluminum stubs and

are coated in gold

Thin films of collodion or

other supporting material on copper grids

Nature of Lenses glass glassglass lenses with dichromatic

mirrorsone electrostatic lens with a few

electromagnetic lenses

one electrostatic lens and a few

electromagnetic lenses

Focusing mechanical mechanicaldigital computer motorized

focusing mechanismelectrical

Electrical i.e. current of the objective lens

coil is changed.

Magnification Adjustments

changing objectives usually 1 objective digitally enhanced electrical

Electrical i.e. changing current of the projector

lens coil

Major Means of Providing

specimen ContrastLight Absorption light scattering or light reflection

laser light with dicromatic mirror concentrated at pinhole

electron scatteringElectron

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 LysoTracker Red

Immunofluorescence

Evans Blue Stain (non-specific red)

Yellow green fluorescing antibody for GLUT 2 transporters

Deconvolution Fluorescence

DNA: BlueMicrotubules: GreenActin 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 MicrographsCultured 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