AMPK Methods and Protocols

microscopy is an effective way to assess the localization of these

proteins following exposure to test substances. Cells are fixed and

stained with antibodies raised against the protein of interest, and its

proximity to the nucleus can be evaluated by co-staining with a

nuclear marker or stain (such as RedDot) (Fig.1). This method

also allows the simultaneous visualization of other proteins, which

is of use when investigating cells that have been infected or trans-

fected with viruses or DNA-encoding tagged proteins, such as con-

stitutively active or dominant negative mutant AMPK. As a

consequence, the infected/transfected cells can be specifically exam-

ined by co-staining of cells, of particular value in cells where trans-

fection/infection efficiency is relatively poor, such as 3T3-L1

adipocytes (Fig.2)[ 12].

The cytokines and chemokines produced as a result of inflam-

matory signalling are then secreted from the cell and can function

in an autocrine, paracrine, and endocrine manner to exacerbate the

pro-inflammatory environment [17, 18]. Here we describe a

method to generate and collect culture medium from cells exposed

to test substances. Levels of secreted proteins can then be measured

quantitatively by an enzyme-linked immunosorbent assay (ELISA).

A consequence of inflammatory chemokine secretion in endo-

thelial cells is the adhesion of monocytes, which is a key step in the

infiltration of the vascular wall by leukocytes that occurs early in

atherogenesis [19]. Infiltrating monocytes in the blood vessel wall

differentiate to macrophages which can become further polarized

toward a pro-inflammatory phenotype and avidly take up modified

Fig. 1Immunofluorescence images obtained following anti-phospho-JNK staining. Wild-type and AMPK-α1/
α2 knockout mouse embryonic fibroblasts (MEFs) [12, 22] were incubated with IL-1β(10 ng/ml) for 15 min
following preincubation for 30 min in the presence or absence of A769662 (100μM) and stained with anti-
phospho-JNK antibodies. RedDot was used to stain the nucleus

308 Sarah J. Mancini and Ian P. Salt