had the protective effect of inhibiting foam cell formation through
the induction of cholesterol efflux [23], suggesting a protective role
for AMPK during atherogenesis. The methodological details of
these experiments and notes pertaining to future AMPK-related
work are explained below.2 Materials
2.1 Isolation
and Culture of Bone
Marrow-Derived
Macrophage (BMDM)
L929-Conditioned Medium- ClassІІbiological safety cabinet (BSCІІ).
- Bead bath (alternatively, hot water bath) set at 37C.
- Humidified 5% CO 2 incubator at 37C.
- 175 cm^2 tissue culture flasks (T175).
- Dulbecco’s modified Eagle’s medium (DMEM): DMEM con-
taining 4.5 g/L glucose and 1 mM sodium pyruvate. - L929 medium: DMEM, 10% (v/v) heat-inactivated fetal
bovine serum (FBS), 100 units/mL penicillin, and 100μg/
mL streptomycin. - Sterile 1phosphate-buffered saline (PBS): 137 mM NaCl,
2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4. - 1trypsin, 0.05% EDTA in PBS.
- L929 murine fibroblast cell line (American Tissue Culture
Collection). - 50 mL sterile conical tubes.
- 0.22μm sterile bottle-top filter.
- Tabletop centrifuge capable of 4C refrigeration for 50 mL
tubes. - Sterile 2 L bottle.
Bone Marrow-Derived Macrophage (BMDM) Isolation- Surgical scissors, forceps, and scalpel.
- 70% (v/v) ethanol diluted in water.
- Ketamine/xylazine cocktail: 150 mg/mL ketamine, 20 mg/
mL xylazine. - 1 mL syringe.
- 23-Gauge needle.
- Sterile 0.5 and 1.5 mL microcentrifuge tube.
- 18-Gauge needle.
- 100 mm tissue culture plates.
- 40μm cell strainers fitted for 50 mL conical tube.
Assessing Macrophage Cholesterol Homeostasis 479