Subfractionation of Liver Cells

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Subfractionation of Liver Cells
Purified samples are required for many types
of analyses. A common way of separating and
purifying cell organelles is to disrupt the
tissue and cell membranes, releasing cell
contents, and use either differential or density
gradient centrifugation to purify the various
cellular components based on their densities. The
photos below show liver cells after gentle
homogenization and liver cell organelles
separated by differential centrifugation after
homogenization in a Teflon-in-glass homogenizer.
Special stains help test the purity of the
fractions and identify organelles.
After 3-4 passes of homogenizer through the
tissue, the cell membranes of most cells are
broken, releasing cell contents. Organelles of
different densities, such as nuclei and
mitochondria, are then separated from the
mixture by centrifugation at varying speeds.
When the homogenizer is passed through minced
liver tissue one time, most of the minced tissue
is broken into small clumps of cells and
individual whole cells.
Special stains can be used to identify cell
organelles.
Methyl green-pyronin (MGP)stains nucleic
acids--RNA stains pink-red and DNA stains
blue-green.
Aceto-orcein (AO) stains chromatin in cell nuclei
dark red.
In the photo below, whole cells and contents of
disrupted cells are present. MGP stains the
cytoplasm of whole cells pink. Blue-green
staining cellular debris in the background
consists mostly of mitochondria. Why do these
stain blue-green?
Shown below are clumps of liver cells stained
with MGP. Cell nuclei appear bluish and
cytoplasmic RNA stains pink.
The photo below shows intact liver cells and bare
nuclei stained with AO.