which must be used as a vehicle control in experiments. Activation
of AMPK by A769662, Compound 991, and AICAR is cell type-
specific and should be optimized for particular cell types prior to
use in functional experiments. Cells can be stimulated with species-
specific pro-inflammatory cytokines IL-1βor TNF-α(10 ng/ml,
incubation time varies depending on application). Care must be
taken to follow the supplier’s instructions concerning storage of
pro-inflammatory cytokines, which are sensitive to freeze-thawing.2.1 Common
Materials to All
Techniques
- Human umbilical vein endothelial cells (HUVECs).
- MV2 complete endothelial cell medium (Promocell).
- Medium 199.
- A769662 stock solution: 50 or 100 mM A769662 in DMSO.
- IL-1βstock solution: 1μg/ml human recombinant IL-1βin
PBS supplemented with 0.1% (w/v) BSA.
2.2 Preparation
of HUVECs for Confocal
Immunofluorescence
Microscopy
- Cell culture plasticware (12-well plates).
- 37C incubator (atmospheric CO 2 ).
- Krebs ringer HEPES buffer (KRH): 119 mM NaCl, 20 mM
HEPES-NaOH, pH 7.4, 5 mM NaHCO 3 , 5 mM KCl, 1.2 mM
MgSO 4 , 1.2 mM NaH 2 PO 4 , 2.5 mM CaCl 2 , and 5 mM
Fig. 3A769662-mediated inhibition of U937 monocytecell adhesion to HUVECs. HUVECs were incubated with
IL-1β(10 ng/ml) for 6 h following preincubation for 30 min in the presence or absence of A769662 (100μM).
Monolayers were washed prior to incubation for 1 h with U937 pro-monocytic cells (2 105 cells/ml). Cells
were fixed and adherent monocytes visualized by microscopy. Scale bar represents 50μm
310 Sarah J. Mancini and Ian P. Salt