AMPK Methods and Protocols

or 3T3-L1 adipocytes and localizing phosphorylated JNK are

found in the Subheading4.

1. Grow cells to confluence on 13 mm diameter coverslips set
   inside 12-well plates (two coverslips per condition, plus an
   extra two coverslips which will become the secondary antibody
   only control).


2. On the day of the experiment, wash cells with serum-free
   Medium 199 (0.5 ml/well) (seeNotes 3and 4 ).


3. Return plates to 37C incubator in appropriate CO 2 atmo-
   sphere and starve for 2 h.


4. After 2 h, remove plates from incubator, aspirate serum-free
   Medium 199, and add 0.5 ml/well KRH buffer. Aspirate and
   replace with another 0.5 ml/well KRH buffer. Transfer plates
   to a 37C incubator at atmospheric CO 2 and incubate for
   approximately 30 min (seeNote 5).


5. Add pharmacological AMPK activator (A769662 at final con-
   centration of 100μM. 1μl of 50 mM stock/well (seeNote 6))
   or equivalent volume of DMSO to control wells (seeNote 7).


6. Return plate(s) to incubator.


7. After 30 min incubation with A769662, remove plate from
   incubator and add pro-inflammatory cytokine (IL-1βat final
   concentration of 10 ng/ml—5μl per well of 1μg/ml stock) to
   appropriate wells.


8. Return plate(s) to incubator and incubate for 15 min.


9. At the end of the assay, remove plate(s) from incubator, aspi-
   rate, and wash cells with PBS.


10. Fix cells for 25 min at room temperature in PBS-PFA (400μl/
    well) (seeNote 8).


11. Aspirate PBS-PFA and quench remaining PFA by washing each
    well twice with PBS-glycine (0.5 ml/well), followed by two
    washes with PBS (0.5 ml/well).


12. Lift coverslips into clean 12 well plate(s) containing
    0.5 ml/well of PBS using hooked forceps and a needle (see
    Notes 9and 10 ).


13. Permeabilize cells with 400μl/well of permeabilization buffer
    for 10 min (seeNote 11).


14. Aspirate permeabilization buffer and wash with PBS.


15. Block nonspecific binding sites with 400μl/well of IF buffer
    (seeNote 12) for 20 min.


16. Cut a section of Whatman 3MM paper, dampen with water,
    and smooth out on bench (seeNote 13). Lay out strip of
    parafilm on top, smoothing out.

312 Sarah J. Mancini and Ian P. Salt