- 7 mL Scintillation vials (or equivalent).
- Scintillation counter (e.g., Tri-Carb 2910 TR, Perkin Elmer).
- Tabletop centrifuge capable of 4C refrigeration.
2.5 In Vivo Reverse
Cholesterol Transport
- ClassІІbiological safety cabinet (BSCІІ).
- Bead bath (hot water bath) set at 37C.
- Humidified 5% CO 2 incubator at 37C.
- 100 mm cell culture dishes.
- 1PBS.
- Recombinant human acLDL (Alfa Aesar).
7.^3 H-Cholesterol (49 Ci/mmol) (Perkin Elmer). - A-769662: 50 mM A-769662 in DMSO (or AMPK activator
of choice). - DMSO.
- LPDS.
- 2 cm cell scraper.
- Trypan blue.
- Hemocytometer.
- 1 mL syringe.
- 23-Gauge needle.
- L929 medium.
- BMDM medium.
- 50 mL sterile conical tube.
- 1.5 mL sterile microfuge tube.
- Container with liquid nitrogen.
- Scintillation cocktail.
- 7 mL scintillation vials (or equivalent).
- Scintillation counter (e.g., Tri-Carb 2910 TR, Perkin Elmer).
3 Methods
3.1 Isolation
and Culture of BMDM
L929-Conditioned Medium- Seed L929 cells at a density of 3.0 103 in 50 mL of L929
medium in a T175 flask. - Incubate cells in a humidified 5% CO 2 incubator at 37C.
- Once cells reach 80–100% confluency, let the flask incubate for
10 days (seeNote 1). - Remove the media (volume will be slightly less than 50 mL) on
day 10 and place in a sterile 50 mL conical tube.
Assessing Macrophage Cholesterol Homeostasis 483