tissue dissociation can be achieved through
mechanical disruption or enzymatic digestion
examples of mechanical digestion
mincing, trituration
examples of enzymatic digestion
trypsin, collagenase, dispase
primary culture
directly isolated from an organism; usually a tedious process
advantage of primary cultures
physiologically relevent
limitations of primary cultures
heterogeneous (cells of varying life stages), finite lifespan, senescence, differentiation
what is the limitation regarding medium for primary cultures?
we dont know the “perfect mix of nutrients, so we pack the culture with good stuff AND serum from the organism
what would happen if a primary culture was contaminated
cells could differentiate, which would create heterogeniality
disadvantage of mechanical disruption
possibility that not all contents will be disrupted
disadvantage of enzymatic digestion
it can alter/disrupt receptor proteins
eukaryotic cell lines
permanently established population of cells derived from a single ancestor (clonal) that can divide indefinitely in laboratory culture
strengths of cell lines
- homogeniality/standardization
- genetic manipulability
- large scale
limitations of cell lines
can differ from original tissue due to genetic drift, altered metabolism, and adaptation to artificial conditions
in cell lines, how can a cell culture change the cells?
a human cell cultures in cow serum will develop different characteristics
differential centrifugation
separate eukaryotic cellular components by centrifugation at increasing levels of speed
in a differential centrifugation, what would the palet consist of when spun at low speed?
nuclear portion
in a differential centrifugation, what would the palet consist of when spun at medium speed?
mitochondria
in a differential centrifugation, what would the palet consist of when spun at high speed?
small organelles
in a differential centrifugation, what would the palet consist of when spun at very high speed?
proteins and macromolecules
in differential centrifugation, after larger/denser components sediment first, sequential spins ca isolate ____
nuclei/mitochondria/membranes/cytosol
cell fractionation by density gradients refine separation by
buoyant density
velocity sedimentation
separation is based on size and shape; a sample is layered on a shallow sucrose gradient and forms distinct, collectable bands
Equilibrium sedimentation
separation based on buoyant density; uses a steep density gradient, particles move until density of particle = density of the solution
which has higher resolution: velocity sedimentation or equilibrium sedimentation
equilibrium sedimentation
