- BMDM medium: DMEM containing 4.5 g/L glucose and
1 mM sodium pyruvate, 10% (v/v) heat-inactivated FBS,
100 units/mL penicillin, and 100μg/mL streptomycin. - L929-conditioned medium.
- Hemocytometer.
- Trypan blue.
- Benchtop microcentrifuge.
- 2 cm cell scraper.
2.2 Cholesterol
Synthesis (^3 H-Acetate
Incorporation)
- Cells of interest: BMDM.
- ClassІІbiological safety cabinet (BSCІІ).
- Bead bath (hot water bath) set at 37C.
- Humidified 5% CO 2 incubator at 37C.
- 12-Well tissue culture plates.
- BMDM medium.
- A-769662: 50 mM A-769662 in dimethyl sulfoxide (DMSO)
(or AMPK activator of choice,seeTable1). - Sodium acetate (500 mM).
9.^3 H-Sodium acetate (75–150 mCi/mmol) (Perkin Elmer). - DMSO.
- Sterile 1PBS.
- Sterile 1.5 mL microfuge tube.
- 1 cm cell scraper.
- Methanol/chloroform (2:1) (v/v).
- Chloroform.
- Double-distilled H 2 O (ddH 2 O).
- Thin-layer chromatography (TLC) plates (silica gel 60).
- Solvent system: heptane/isopropyl ether/acetic acid (60:40:3)
(v/v/v). - Cholesterol standard.
- Hamilton syringe.
- Nitrogen gas.
- Iodine crystals.
- Flat-edged razor blade.
- Weigh paper.
- Scintillation cocktail.
- 7 mL scintillation vials (or equivalent).
- Scintillation counter (e.g., Tri-Carb 2910 TR, Perkin Elmer).
- Bicinchoninic acid assay (BCA) protein determination kit
(or equivalent assay).
480 Nicholas D. LeBlond and Morgan D. Fullerton