Measurement of Autophagy in Cells and Tissues - Protein Misfolding and Cellular Stress in Disease and Aging - page 203

3. If using Hanks' balanced salt solution (HBSS with calcium
and magnesium) instead of EBSS or KRB as starvation
medium, HBSS should be buffered by adding 10 mM
HEPES-NaOH. Calcium and magnesium in HBSS are essen-
tial. When cells are incubated in HBSS, it may be better to
incubate them at 37°C without control of CO 2 .
4. PBS is an unsuitable solution for autophagy induction. PBS is too
severe for cells and cultured tissues to incubate for long times, as
it contains no magnesium, calcium, glucose, or amino acids.
5. One frequently asked question involves the separation and
recognition of LC3-I and LC3-II. The ratio of acrylamide: meth-
ylene-bisacrylamide (29:1 (w/w)) is good for separation and
detection of LC3-I and LC3-II on SDS-PAGE. While 30-40%
acrylamide solution (30:1 (w/w) acrylamide:methylene-
bisacrylamide) is available commercially, the separation of
LC3-I and LC3-II is poor. A final concentration of acrylam-
ide of 12.5% is good. Higher concentrations (>15%) are not
suitable for separation of LC3-I and LC3-II. When using a
ready-made gel for Western blotting, a NuPAGE system
(4-12% or 12% Bis-Tris gel) using MES buffer (Invitrogen)
works well.
6. For good separation between LC3-I and LC3-II, it is better
to run phenol red (New England Biolabs's prestained marker)
through a gel and to stop running just before bromophenol
blue reaches the bottom of the gel.
7. All secondary antibodies conjugated to HRP were purchased
from Jackson ImmunoResearch (West Grove, PA). If the
chemiluminescence signal from HRP using other company's
secondary antibody is insufficient to recognize a specific sig-
nal for the antibody, it is better to use the antibody from
Jackson ImmunoResearch.
8. Image-IT FX markedly reduces background fluorescence
from anti-rabbit, mouse, and chicken IgG conjugated to
Alexa Fluor. IF-blocking solution (2% (w/v) BSA, 5% (v/v)
normal goat serum, 20 mM Tris-HCl, pH 7.5, 150 mM
NaCl) can be used as the blocking solution instead of
Image-IT FX, but background signal is increased.
9. 0.25 M sucrose, 2% Triton X-100, and 1% SDS do not per-
turb the measurement of protein concentration using a BCA
protein assay kit.
10. This method can be applied for cultured cells. For tissue
preparations, please refer to Waguri and Komatsu ( 34 ).
11. Washing medium before fixation is usually unnecessary and
may cause some morphological changes at the electron micro-
scopic level. Some researchers adopted a method where cells