Tools of Cell Biology


22 Φεβ 2014 (πριν από 7 χρόνια και 8 μήνες)

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Tools of Cell Biology

BIO 224

Intro to Molecular and Cell Biology

Other Tools of Cell Biology

Microscopy alone is not sufficient to define all
functions of eukaryotic cell parts

Subcellular organelles need to be isolated and
subjected to biochemical studies

Various tools and methods available to
achieve fractionation of subcellular

Often rely on centrifugation methods

Centrifugation Methods

Differential centrifugation

Developed in 1940s an d1950s

Separates cellular components based on size
and density

Must be carefully undertaken to avoid
destruction of components of interest

Stepwise process

Differential Centrifugation

Disruption of plasma membrane first step

Must avoid destroying cellular components

Sonication, grinding, high
speed blending


to separate
fractionate components

Rotation speeds greater than 100K rpm, up to 500K
times gravity

Sediments components at bottom of tube

Sedimentation rate depends on size and density

Differential Centrifugation

Suspension initially spun at low speed to
pellet unbroken cells and nuclei

Supernatant spun at higher speed to pellet
mitochondria, chloroplasts, peroxisomes,

Recentrifugation at higher speed pellets
plasma membrane and ER fragments

Fourth higher speed centrifugation leaves only
cytosol in supernatant

Centrifugation Methods

gradient centrifugation

can be used to
further purify preparations from differential

Organelles are separated through gradient of dense
substance like sucrose

Velocity centrifugation

places material on top of sucrose

Different size particles sediment through gradient at varying rates

Move as discrete bands

Fractions may be collected following centrifugation to separate
organelles of similar size (mitochondria, lysosomes, peroxisomes)

Gradient Centrifugation

Equilibrium centrifugation

Used to separate components based on buoyant

Independent of shape and size

Sample centrifuged in high concentration of
sucrose or cesium chloride solution

Samples spun until
equilibrium position


Where buoyant density equals surrounding solution

Useful to separate different membrane types and
macromolecules labeled with different isotopes

Cell Culture

Variety of cells can be grown in culture

Allow for studies of cell growth and
differentiation, and genetic manipulations

Started by placing piece of tissue into
suspension of component cells

Suspension added to dish containing
appropriate media

Most cell types grow on plastic surfaces of
culture dishes

Cell Culture

Embryos and tumors often used to start

Embryo fibroblasts one of most commonly
studied animal cell types

Many cell types can be grown under optimum

Culture media for animal cells more complex
than for microorganisms

Early media had undefined components

Plasma, serum, embryo extracts

Cell Culture

1955 Harry Eagle described first defined
media that supported animal cell growth

Animal cells have complex requirements

All biochemical needs must be supplied by
growth medium for cell survival

Biochemical studies have allowed for
development of serum
free media

Cell Culture

Cultures established from tissues are

Primary culture cells grow until they cover dish surface

Can be removed from the dish and replated at lower density to
form secondary cultures

Process can be repeated many times

Most normal cells cannot be grown indefinitely

Normal human fibroblasts can be cultured 50 to 100
population doublings

Embryonic stem cells and tumor
derived cells can be
maintained indefinitely and are referred to as

Cell Culture

Division time around is 20 hours under
optimal conditions for most normal cells

Experiments with animal cells more difficult
and take longer than with microbes

Animal cells cultures have been very
necessary for experiments leading to
understanding of their structure and function

Cell Culture

Plant cells can also be cultured

Mass of undifferentiated plant cells is called a

Many plant cells can form cell types and
tissues needed to regenerate entire plant

Undifferentiated cells can be induced to form
many cell types

Easy for genetic modification of plants


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