AMPK Methods and Protocols

2.3 Acetylated
Low-Density
Lipoprotein (acLDL)
Uptake

1. Cells of interest: BMDM.


2. ClassІІbiological safety cabinet (BSCІІ).


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


4. 37C and 5% CO 2 cell incubator.


5. 24-Well tissue culture plates.


6. BMDM medium.


7. 1PBS.


8. A-769662:100μM A-769662 in DMSO (or AMPK activator
   of choice,seeTable1).


9. DMSO.


10. Recombinant human low-density lipoprotein (LDL) (Alfa
    Aesar).


11. Recombinant human acLDL (Alfa Aesar).


12. 1,1^0 -Dioctadecyl-3,3,3^0 ,3^0 -tetramethylindocarbocyanine (Dil)-
    acLDL (Alfa Aesar).


13. Isopropyl alcohol.


14. Parafilm.

Table 1
Commonly used compounds for activating macrophage AMPK

Compound name Mechanism of action

Working

concentration Reference

Metformin "AMP/ATP 0.2–2 mM [24]

Berberine "AMP/ATP 5 μM[25]

Resveratrol "AMP/ATP 0.1–10μM[26]

Troglitazone "AMP/ATP 10 μM[27]

Phenformin "AMP/ATP 100 μM[28]

Curcumin "AMP/ATP 0.5–10μM[29]

Thienopyridone (A-769662) Allosteric activation/

inhibition of

dephosphorylation

100 μM[14, 23]

Salicylate Allosteric activation 3 mM [23]

5-Aminoimidazole-4-

carboxamide ribonucleotide

(AICAR)

AMP mimetic 0.1–2.5 mM [18]

Benzimidazole (Compound 911) Allosteric activation Presumed to be

0.1–5μM

Yet to be tested in

macrophages

Compound 13 Allosteric activation Presumed to be

10–100μM

Yet to be tested in

macrophages

Assessing Macrophage Cholesterol Homeostasis 481