of AMPK for the induction of autophagy in response to a stimulus
of interest.
In the following we describe the protocols that have been used
in our lab to measure and evaluate autophagy with the particular
emphasis on the role of AMPK in this process. We present cell-
based systems to study AMPK-dependent autophagy by biochemi-
cal assays using immunoblotting and immunofluorescence as inde-
pendent approaches.2 Materials
Store all reagents and solutions at room temperature or according
to the manufacturer’s instruction unless stated otherwise. Always
use double-distilled and autoclaved H 2 O.2.1 Cell Lines 1. U2OS: human bone osteosarcoma cell line (ATCC®
HTB-96™).
- NIH3T3: immortalized murine fibroblasts (ATCC®
CRL-1658™). - NIH3T3 mCherry-EGFP-LC3: NIH3T3 cells retrovirally
transduced with pBABE-puro mCherry-EGFP-LC3 (Addgene
#22418) [28–30].
2.2 Cell Culture 1. DMEM high glucose: Dulbecco’s Modified Eagle Medium
(DMEM), 4.5 g/L glucose, 4 mML-glutamine, 10% (v/v)
heat-inactivated fetal calf serum (FCS), 100μg/ml penicillin/
streptomycin (P/S). Store at 4C.
- Glucose-free DMEM: DMEM without glucose, 4 mML-glu-
tamine, 10% (v/v) heat-inactivated FCS, 100μg/ml P/S. Store
at 4C. - Puromycin: 2 mg/ml puromycin in DMSO. Store at 20 C.
- Phosphate-buffered saline (PBS) (pH 7.3): 140 mM NaCl,
2.6 mM KCl, 2 mM Na 2 HPO 4 , 1.45 mM KH 2 PO 4. - Opti-MEM®medium. Store at 4C.
- AICAR: 150 mM aminoimidazole-4-carboxamide ribonucleo-
tide (AICAR) in H 2 O. Store at 20 C; use within 1 month. - A-769662: 50 mM A-769662 in DMSO. Store at 20 C; use
within 1 month. - Baf. A1: 200 mM bafilomycin A1 in ethanol. Store at 80 C.
- Tissue culture dishes: diameter 6 or 10 cm.
- Glass coverslips: diameter 18 mm.
- Cell-counter instrument or Neubauer chamber.
- CO 2 incubator: Set to 5% CO 2 and 37C.
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