than two coverslips should be mounted in parallel since the
Mowiol spots would dry out otherwise.
- Cells need to be tested for mycoplasma, since these pathogens
induce autophagy and thus distort the results.
- For experiments investigating autophagy, the cells should not
have a high passage number (<15), because the regulation of
autophagy and their sensitivity to stimuli change over time.
- When evaluating the induction of autophagy, a control trans-
fection with empty vectors should always be included, since
transfection already induces autophagy to some extent.
- If the cell density is too high or the medium is exhausted, the
resulting energy deprivation induces autophagy and falsifies the
results.
- It works best to place the coverslip in a dish before adding the
medium. Then press the coverslip onto the dish with a plastic
pipette tip in order to fixate but not break it.
- In experiments using the NIH3T3 cells stably expressing the
tandem-tagged LC3 construct, the puromycin selection needs
to be abolished already one passage prior to seeding for the
planned experiment, since puromycin is known to interfere
with autophagy [31–33].
- In the unstimulated cells, the vehicle (DMSO, ethanol, etc.)
should be used as a control to exclude effects on the induction
of autophagy.
- If the induction of autophagy is evaluated by the means of the
conversion of LC3, the stimulation with AICAR, A-769662,
or glucose starvation needs to be applied for longer time
periods or overnight. After treatment for 1–2 h, which, e.g.,
is sufficient for EBSS starvation, only phosphorylation of direct
substrates of AMPK can be assessed [2, 34].
- The stimulation with AICAR, A-769662, or glucose starvation
is more specific than, e.g., the broader EBSS starvation and
allows the evaluation of AMPK-dependent induction of autop-
hagy in particular [1–4, 35].
- A longer treatment with bafilomycin A1 leads to a saturation of
the LC3-II amount and induces cell death [36, 37].
- The double-tagged mCherry-EGFP-LC3 fusion protein allows
the discrimination between autophagosomes and autolyso-
somes since EGFP loses its fluorescence in the acidic environ-
ment of the autolysosomes resulting in an only red signal while
autophagosomes are labeled red and green and thus appear
yellow in a merged picture [30, 38].
- If only an immunoblot from whole cell lysates is performed, a
6 cm dish will usually be sufficient and saves siRNA.
Autophagy 387
