Biomedical Engineering Reference
In-Depth Information
LC3-lipidation blocking buffer for LI-COR quantitative infrared
fl uorescence Western blots: 5 % BSA in PBS.
LC3-lipidation blocking buffer for ECL: 5 % skimmed milk, 0.1 %
Tween-20, PBS.
Washing buffer: PBST (PBS, 0.1 % Tween 20).
2.3 Autophagy
Inducers
and Inhibitors
Earle's balanced salt solution (EBSS): 5.56 mM
D
-glucose,
123.08 mM NaCl, 5.37 mM KCl, 1.82 mM CaCl
2
, 0.81 mM
MgSO
4
, 0.99 mM Na
2
HPO
4
, 13.10 mM NaHCO
3
.
Rapamycin: 11
M in complete cell culture medium.
Torin 1: 100 nM fi nal in complete cell culture medium.
Bafi lomycin A1: 100 nM fi nal in complete cell culture medium or
EBSS (Calbiochem).
Wortmannin: 100 nM fi nal in complete cell culture medium or
EBSS (Calbiochem).
ʼ
3
Methods
All steps were performed at room temperature, and solutions are
kept at room temperature unless otherwise indicated. We routinely
use HEK293A cells as they respond robustly to amino acid starva-
tion. Note: all PBS is CMF-PBS, i.e., calcium- and magnesium-free:
3.1 Immuno-
fl uorescence
Microscopy
1. Prepare a 12-well plate by adding cleaned, microscopy quality
13 mm round glass coverslips. Coat with poly-
D
-lysine if cells
are not very adherent (
see
Note 2
). If rabbit anti-LC3 is being
used with the mouse anti-WIPI2 antibody, then only one set of
coverslips is needed. If both mouse anti-LC3 and mouse anti-
WIPI2 are used, then set up two separate sets of coverslips. For
additional information about autophagosome maturation, use
LAMP1 antibodies in combination with LC3 (
see
Note 3
).
2. Plate cells in a 12-well dish at a density which will yield a
60-70 % confl uency at the point of fi xation, e.g., plate 5-7 × 10
4
HEK293A cells per well for fi xation the following day.
Return to incubator and incubate overnight.
3. Perform experimental manipulations or starvation experiment
as appropriate. All medium should be pre-warmed to 37 °C. To
prepare samples for LC3 staining under amino acid starvation,
wash ½ of the coverslips twice with 1 ml complete cell culture
medium (fed), and wash the other ½ twice with 1 ml EBSS.
4. To dishes washed with growth medium, add:
Fed: add 1 ml growth medium.
Fed plus BafA: add 1 ml growth medium plus 100 nM bafi lo-
mycin A1.
