Measurement of Autophagy in Cells and Tissues - Protein Misfolding and Cellular Stress in Disease and Aging - page 198

3. Methods

To monitor autophagy, separation, and recognition of endog-
enous LC3-I (cytosolic form) and LC3-II (membrane-bound
form) are critical in immunoblotting. Anti-LC3 antibodies, which
are useful to recognize endogenous LC3-I and LC3-II, are now
commercially available (Table 2 ). The listed anti-LC3 antibodies
are also available for immunofluorescence analysis. There are
some key points for sample preparation for SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) and immunofluorescence analy-
sis. LC3-II (LC3-phosphatidylethanolamine conjugate) only dis-
solves partially in 1% Triton X-100, which is usually used for cell
lysis due to its hydrophobicity. Therefore, when cells and tissues
are lysed for SDS-PAGE, 2% Triton X-100 or 1% SDS should be
used as the detergent. In contrast, structures of autophagosomes
and autolysosomes are sensitive to MeOH- and Triton X-100
treatments. Therefore, for investigation of LC3 on autophago-
somes and autolysosomes by immunofluorescence microscopy,
digitonin or saponin is suitable for cell permeabilization.
There are two types of autophagy in mammals: inducible
autophagy and constitutive autophagy. Inducible autophagy
occurs under starvation conditions, and is observed mainly in the
liver and muscles. This type of autophagy can be monitored by
determining the levels of LC3-II and dephosphorylation of mTor-
signaling pathway. On investigation of autophagic responses in
tissues, the conditions for feeding and fasting should be consid-
ered. In general, after feeding, autophagy in the liver is suppressed
to store nutrients in the liver. Autophagy in the liver is re-induced
again several hours after feeding. When fasting continues for over
6 h, autophagy in the skeletal muscles occurs. Thereafter,
autophagy in the heart occurs when fasting lasts longer.
Constitutive autophagy is the basal level of autophagy and
functions mainly in maintenance of cells, especially neurons,
and tissue-dependent physiological functions. Neuronal cell death
by ablation of autophagy in the brain is caused by constitutive
autophagy. In contrast to inducible autophagy, there is little increase
of LC3-II during constitutive autophagy. However, as autophagic
insufficiency even in these tissues leads to an increase of p62 and
ubiquitin-positive aggregates, it is better to monitor p62 and ubiq-
uitin by Western blotting, immunohistochemistry, and immuno-
electron microscopy for estimation of autophagic insufficiency.

1. Pre-warmed KRB or EBSS buffer was prepared to investigate
the autophagic responses of cells and cultured tissues under
starvation conditions (see Note 3 when using HBSS instead).
When lysosomal degradation of LC3-II is suspected, 1:1,000
dilutions of the stock solutions of E64d and pepstatin A

3.1. Cell Culture and
Preparation of Cell
Lysate