that AMPK is very sensitive to stress and can be readily phosphory-
lated and activated during harvesting and extraction of cells or
tissues. If great care is not taken over the methods used for cell or
tissue harvesting, this can lead to many misleading artifacts. The key
to success is to achieve rapid cooling of the cells or tissues. This was
first realized by Zammit and coworkers when they prepared ER
membranes for assays of the downstream target of AMPK,
HMG-CoA reductase, which led to the development of a technique
known as “cold-clamping” [9]. It was reinforced when our labora-
tory found that freeze-clamping was necessary to preserve the
phosphorylation status of another AMPK target, acetyl-CoA car-
boxylase, during extraction from rat liver [10]. When sampling
human muscle, which is normally achieved using needle biopsies,
the biopsies are frozen in liquid nitrogen as soon as they are taken
[11]. We describe below (Subheading2.1) our standard method
for harvesting cells that show good adherence to culture plates
following growth in two-dimensional culture, which we have devel-
oped over many years. We also describe (Subheading2.2) a method
we have developed for harvesting cultured cytotoxic T lympho-
cytes, which grow in suspension and do not adhere to culture
plates. These methods can be adapted to other cell types, according
to whether or not the cells are adherent. However, when harvesting
AMPK from any tissue or cell type, it is necessary to think carefully
about whether the method used could be leading to artificial
activation, eitherpostmortemwhen using animal tissues or during
harvesting when using cultured cells.2 Materials
Prepare all solutions with ultrapure water. Reagents are stored at
room temperature unless indicated otherwise.2.1 Methods
of Plating and Treating
Adherent Cells
- Laminar flow tissue culture cabinet.
- Tissue culture incubator.
- Cells in 6 cm plates.
- Compounds to activate AMPK, e.g., direct activators such as
A.769662 or indirect activators such as phenformin or
berberine. - Pipette and tips.
- Culture medium.
- Fetal bovine serum (FBS).
- Penicillin-streptomycin solution.
- Cell scraper.
- Aspirator.
- Plastic pastette.
242 Simon A. Hawley et al.