AMPK Methods and Protocols

glucose. A 10 solution without CaCl 2 or glucose can be

prepared and stored at 4C. CaCl 2 and glucose can be added

to a 1solution on day of use (seeNote 1).

4. Phosphate-buffered saline (PBS).


5. PBS-glycine: PBS, 20 mM glycine.


6. PBS-PFA: PBS, 3% (w/v) paraformaldehyde (seeNote 2).


7. Permeabilization buffer: PBS, 2% (w/v) BSA, 0.1% (v/v) Tri-
   ton-X-100, 20 mM glycine.


8. IF buffer: PBS, 2% (w/v) BSA, 20 mM glycine, 0.1% (v/v) goat
   serum.


9. Whatman 3MM paper.


10. Parafilm.


11. Hooked forceps.


12. 21-Gauge needle.


13. 13 mm coverslips and microscope slides.


14. Rabbit anti-NFκB (p65) antibodies.


15. Alexa Fluor®488-linked goat anti-rabbit antibodies.


16. RedDot far-red nuclear stain (Biotium).


17. Immunomount.


18. Confocal fluorescence microscope.

2.3 Preparation
of HUVEC-Conditioned
Medium for Cytokine/
Chemokine Analysis

1. Cell culture plasticware (6-well plates).


2. Human IL-6 or MCP-1 ELISA kit (R&D Quantikine kits).


3. Spectrophotometer capable of measuringA450/570in 96-well
   ELISA plates.

2.4 Analysis of U937
Cell Adhesion
to HUVECs

1. Cell culture plasticware (24-well plates).


2. U937 cells.


3. RPMI 1640 medium.


4. DMEM.


5. PBS-sucrose-PFA: PBS, 4% (w/v) paraformaldehyde (PFA),
   5% (w/v) sucrose, pH 7.2 (seeNote 2).


6. Light microscope with a 5objective.

3 Methods

3.1 Preparation
of HUVECs for Confocal
Immunofluorescence
Microscopy to Assess
Nuclear Localization
of NFκB

Here we describe methods to investigate the effect of AMPK

activation on the subcellular localization of NFκB p65. The method

varies slightly depending on cell type. The method described below

has been successfully utilized in HUVECs, while the alternative

steps required for fixing and staining mouse embryonic fibroblasts

Roles in Inflammation 311