Tools of Cell Biology

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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
components


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


Suspension
ultracentrifuged

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,
lysosomes


Recentrifugation at higher speed pellets
plasma membrane and ER fragments


Fourth higher speed centrifugation leaves only
cytosol in supernatant

Centrifugation Methods


Density
-
gradient centrifugation

can be used to
further purify preparations from differential
centrifugation


Organelles are separated through gradient of dense
substance like sucrose


Velocity centrifugation

places material on top of sucrose
gradient


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)

Density
-
Gradient Centrifugation


Equilibrium centrifugation


Used to separate components based on buoyant
density


Independent of shape and size


Sample centrifuged in high concentration of
sucrose or cesium chloride solution


Samples spun until
equilibrium position

reached


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
conditions


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
cultures


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
lines


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
callus


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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