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and (iv) treatment with statins, which inhibit HMG-CoA reductase activity, thus
blocking cholesterol biosynthesis ( Allen et al., 2007 ). These techniques are usually
combined with protein extraction or cellular imaging studies, providing relevant in-
formation about protein interactions with lipid rafts. Due to their relatively easy im-
plementation and short incubation times, cyclodextrin and cholesterol oxidase
treatment are the most widely used methods to disrupt rafts.
6.1.1.1 Methyl- b -cyclodextrin
Cyclodextrins are water-soluble oligosides composed of six to eight
(1-4)-
glucopyranose units arranged in a closed ring structure. Their external surface is
hydrophilic, while their inner cavity is hydrophobic. These molecules enhance the
solubility of hydrophobic compounds (e.g., cholesterol) by encapsulating them in
their inner cavity ( Gimpl & Gehrig-Burger, 2007; Zidovetzki & Levitan, 2007 ).
Methyl-
b
C), which contains seven methylated oligoside units,
has the highest affinity for cholesterol, being also the most selective and efficient
in extracting it from cells. Depending on concentration, incubation time, tempera-
ture, and cell type, the amount of cholesterol extracted from the cells by these
molecules is variable. For lipid raft disruption studies cells are typically incubated
with 5-10 mM of M
b
-cyclodextrin (M
b
C for 0.5-2 h at 37 C. This results in cholesterol
extraction percentages between 30 and 60% of the cell's total free cholesterol
( Zidovetzki & Levitan, 2007 ). However, these levels of extraction submit cells to
a stress situation, which is further aggravated by the higher permeability of their
cholesterol-poor membranes ( Gimpl & Gehrig-Burger, 2007; Zidovetzki &
Levitan, 2007 ). Moreover, other membrane components might be also extracted
from the cells by cyclodextrin. Therefore, a careful selection of the experimental
conditions and rigorous control experiments should be done. A common approach
to assess the existence of artifacts in these types of experiments is to add cholesterol
back to drug-treated cells. If the effects of the drug treatment are reversed, then it is
likely that the experimental observations are not being affected by cholesterol extrac-
tion pleiotropic effects. This can be done by loading cyclodextrins with cholesterol in
vitro and presenting them to drug-treated cells to replenish their cholesterol levels
( Allen et al., 2007 ).
b
6.1.1.2 Cholesterol oxidase
Cholesterol oxidase (EC 1.1.3.6) is a microbial flavin adenine dinucleotide-
containing enzyme (flavoenzyme), belonging to the oxidoreductase family ( Gimpl &
Gehrig-Burger, 2007 ). It catalyzes cholesterol oxidation to cholest-5-en-3-one
and subsequent isomerization to cholest-4-en-3-one, which does not have the same
membrane-ordering properties as cholesterol ( Castro, Silva, Fedorov, de Almeida,
& Prieto, 2009 ). Since its first application to track cholesterol cellular localization
and to probe cell membrane heterogeneities ( Lange, 1992 ), this enzyme has be-
come a general tool in cell membrane organization studies. Depending on the cell
type, raft disruption is normally achieved by incubating cells with 0.5-2 U/mL of
cholesterol oxidase in a serum-free medium for 0.5-2 h at 37 C( Cahuzac et al.,
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