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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.
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.
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.
Liver cell nuclei (arrows) stained with
aceto-orcein
Clumps of unstained liver cells after one
pass through homogenizer
Special stains can be used to identify cell organelles.
Methyl green-pyronin (MGP)stains nucleic acids--RNA stains pink-red and DNA stains bluegreen.
Shown below are clumps of liver
cells stained with MGP. Cell
nuclei appear bluish and
cytoplasmic RNA stains pink.
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?
Aceto-orcein (AO) stains
chromatin in cell nuclei dark red.
The photo below shows intact liver
cells and bare nuclei stained with
AO.