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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