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

• MOLECULAR CELL BIOLOGY

• SIXTH EDITION

CHAPTER 9
Visualizing, Fractionating,
and Culturing Cells
© 2008 W.
Copyright H.©Freeman
2008 andand
W. H. Freeman Company
Company
Fluorescence
Alpha tubulin (green)
Actin (red)
DNA (blue)
Golgi (yellow)
Mitochondria (purple)
Cell Isolation, Culture and
Differentiation
 (a)Humancells
Phase Phase
PhaseII III

Proteases
Cellstrain
• trypsin
Growth
Divalent cation chelator
rateof
• EDTA culture
Senescence Cell
• 50 times senescence

Cell Strain: line of cell that have a L

25 50
limited life span
Cellgenerations
Cell line: line of cells that are
immortal (b)Mousecells

Initialloss Emergenceof
ofgrowth immortal
potential variant
Growth (cellline)

rateof
Senescence
culture

30 60
Daysafterinitiationofculture
Figure9-31
MolecularCellBiology,SixthEdition
©2008W.H.FreemanandCompany
 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
Injectmouse
withantigenX

•Myeloma cell: Immortal

•Mouse spleen cell: mortal
•Viral glycoproteins/polyethylene glycol
•Grown in media that does not allow
unfused cells to grow (mutations in Mutantmouse
myelomacells
Mousespleencells;
somecells(red)make
metabolic pathways). Fused cells have unabletogrow
inHATmedium
antibodytoantigenX
Mixand O' о'
ability to grow due to sharing of genetic fusecellso. O.

material that was deleted from other cells. 2 Transferto

HATmedium

Unfusedcells
(000)die
Fusedcells
(00)grow

3 Culturesinglecells
inseparatewells

TesteachwellforantibodytoantigenX
Figure9-35
MolecularCellBiology,SixthEdition
©2008W.H.FreemanandCompany
 Left: Bright Field
Middle: Differential interference contrast
Right: Phase Contrast

Figure9-11
MolecularCellBiology,SixthEdition
©2008W.H.FreemanandCompany
Epifluorescencelightpath

Dichroicmirror

000

Specimen

Figure9-10d
MolecularCellBiology,SixthEdition
©2008W.H.FreemanandCompany
The table below compares the different types of microscope:
http://universe-review.ca/R11-13-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
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 glass 1 electrostatic + a few em. lenses 1 electrostatic + a few em. lenses

Focusing mechanical current in the objective lens coil current in the objective lens coil

Magnification Adjustments changing objectives current in the projector lens coil current in the projector lens coil

Specimen Contrast by light absorption by electron scattering by electron scattering

 Transmission
Compound Dissection or Stereoscope Confocal Microscope Scanning Electron Microscope (SEM) Electron
Microscope (TEM)

TEM is electron
illuminated. This
Compound microscopes are light SEM use electron illumination. The image
A dissection microscope is light This microscope uses a laser light. This gives a 2-D view.
illuminated. The image seen with this type is seen in 3-D. It has high magnification
illuminated. The image that appears is light is used because of the wavelength. Thin slices of
of microscope is two dimensional. This and high resolution. The specimen is
three dimensional. It is used for dissection Laser light scan across the specimen with specimen are
Description microscope is the most commonly used. coated in gold and the electrons bounce off
to get a better look at the larger specimen. the aid of scanning mirrors. Then image is obtained. The
You can view individual cells, even living to give you and exterior view of the
You cannot see individual cells because it then placed on a digital computer screen electron beams pass
ones. It has high magnification. However, specimen. The pictures are in black and
has a low magnification. for analyzing. through this. It has
it has a low resolution. white.
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

visible light visible light laser light electrons electrons
Image Formation

Medium air air air vacuum vacuum

Thin films of
Mounted on aluminum stubs and are collodion or other
Specimen mounting glass slides none glass slides with dyed samples
coated in gold supporting material
on copper grids

one electrostatic lens

one electrostatic lens with a few and a few
Nature of Lenses glass glass glass lenses with dichromatic mirrors
electromagnetic lenses electromagnetic
lenses

Electrical i.e. current

digital computer motorized focusing
Focusing mechanical mechanical electrical of the objective lens
mechanism
coil is changed.

Electrical i.e.
Magnification changing current of
changing objectives usually 1 objective digitally enhanced electrical
Adjustments the projector lens
coil

Major Means of
laser light with dicromatic mirror
Providing specimen Light Absorption light scattering or light reflection electron scattering Electron scattering
concentrated at pinhole
Contrast
Specimen Specimen
holder block BlockSpecimen
Knife

Knife

Cut
section

Microtome

Sections\

Copper
Microscopeslide mesh
grid
Figure9-16
MolecularCellBiology,SixthEdition
©2008W.H.FreemanandCompany
Fura-2 Calcium sensitiveGreen: High Ca
flourochrome Blue: Low Ca
Why are Calcium concentration moving?
Lysosomes and
Mitochondria
Green: Mitochondria
stained with Mitotracker
Green
Red: Lysosomes stained N
with LysoTracker Red

Figure9-3
MolecularCellBiology,SixthEdition
©2008W.H.FreemanandCompany
Immunofluorescence

Evans Blue Stain (non-specific red)

Yellow green fluorescing antibody for GLUT 2 transporters
Deconvolution Fluorescence

DNA: Blue
Microtubules: Green
Actin microfilaments: Red
 Peroxisomes

0.5um
Figure9-21b
MolecularCellBiology,SixthEdition
©2008W.H.FreemanandCompany
 (b)

(c)

Figure9-22bc
MolecularCellBiology,SixthEdition
©2008W.H.FreemanandCompany
animalcell IPlasmamembrane
2Mitochondria
3Lysosomes
4Nuclearenvelope

5Nucleolus

6Nucleus

7Smoothendoplasmicreticulum(SER]
8Roughendoplasmicreticulum(ER)

048pdddEe 9Golgicomplex
10Secretoryvesicles
plantcell
11Peroxisomes
12Cytoskeletalfibers
13Microvilli
14Cellwall
15Vacuole
16Chloroplast

Figure9-1
MolecularCellBiology,SixthEdition
©2008W.H.FreemanandCompany
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