AMPK Methods and Protocols

15. Aluminum foil.


16. 1.5 mL microfuge tube.


17. Fluorometric plate reader (e.g., Biotek Synergy H1 plate
    reader).


18. Microplate for fluorescence based assays (96-well).


19. Scientific rocker.

2.4 Cholesterol
Efflux

1. Cells of interest: BMDM.


2. ClassІІbiological safety cabinet (BSCІІ).


3. Bead bath (hot water bath) set at 37C.


4. Humidified 5% CO 2 incubator at 37C.


5. 24-Well tissue culture plates.


6. 1PBS.


7. Recombinant human acLDL (Alfa Aesar).
   8.^3 H-Cholesterol (49 Ci/mmol) (Perkin Elmer).


8. Bovine serum albumin (BSA), fatty acid-free, low endotoxin
   (Sigma).


9. A-769662:50 mM A-769662 in DMSO (or AMPK activator of
   choice,seeTable1).


10. DMSO.


11. Recombinant human high-density lipoprotein (HDL) (Alfa
    Aesar).


12. Recombinant human apolipoprotein A-І (apoA-І) (Alfa
    Aesar).


13. Lipoprotein-deficient fetal bovine serum (LPDS) (Alfa Aesar).


14. BMDM medium.


15. Lipid loading medium: DMEM containing 4.5 g/L glucose
    and 1 mM sodium pyruvate, 5% (v/v) heat-inactivated LPDS,
    50 μg/mL acLDL.


16. Equilibration medium: DMEM containing 4.5 g/L glucose
    and 1 mM sodium pyruvate, 5% (v/v) heat-inactivated LPDS,
    0.2% BSA fatty acid free, low endotoxin (w/v).


17. HDL efflux medium: Equilibration medium supplemented
    with 50μg/mL HDL.


18. ApoA-Іefflux medium: Equilibration medium supplemented
    with 5μg/mL ApoA-І.


19. Basal efflux medium: DMEM containing 4.5 g/L glucose and
    1 mM sodium pyruvate, 5% (v/v) heat-inactivated LPDS, 0.2%
    BSA fatty acid free, low endotoxin (w/v).


20. NaOH: 0.1 M NaOH.


21. Scintillation cocktail.

482 Nicholas D. LeBlond and Morgan D. Fullerton