- Incubate cells in a humidified 5% CO 2 incubator at 37C for
30 h. - Cells can be treated simultaneously with AMPK activators (see
Note 22). - Remove medium and wash twice with 1PBS.
- Add 2–3 mL of BMDM medium to each plate.
- Gently scrape cells and place in 50 mL conical tube.
- Dilute each genotype or treatment cell suspension 1:1 in trypan
blue, and count the number of live cells using a
hemocytometer. - Inject 8 106 cells into the intraperitoneal cavity (seeNote 23)
of a wild-type mouse (seeNote 24). - Place at least one aliquot of cells (8 106 cells) from each
experimental group into a 7 mL scintillation vial containing
4 mL of scintillation cocktail to determine the initial radioac-
tivity of the injected cells. - Singly house mice after the injection, preferably in a cage with
wire bottom or without bedding (for collection of feces). - Sample radioactivity in the serum by taking blood at 12 and
24 h post injection from the tail vein, and centrifuge at
4000 g for 5 min. Add 10μL of aliquot of serum to
scintillation vial for LSC. - At 48 h post injection, sacrifice mice according to institutional
recommendations (either CO 2 or a lethal anesthetic overdose
to keep vasculature intact). Perform a cardiac puncture by
opening the plural cavity to collect terminal (48 h) blood
sample for serum. - Perfuse the entire circulation with 10 mL of 1PBS via the left
ventricle by making a cut in the right atrium. - Remove the liver and place into a 15 mL tube.
- Digest in 10 mL of 0.1 M NaOH at 60C for 3 h.
- Centrifuge debris at 14,000gfor 10 min and count an
aliquot of the supernatant via LSC. - Collect all fecal pellets produced by the mouse over the course
of the 48 h experiment into a 50 mL tube. - Digest the feces in excess volume of 0.1 M NaOH at 60C for
3 h, followed by a 14,000gspin for 10 min. - Remove an aliquot of the supernatant to a scintillation vial and
determine radioactivity via LSC. - Serum results are expressed as a percent of the initial radioac-
tivity per mL of serum, whereas liver and feces results are
expressed as a percent of the initial radioactivity.
Assessing Macrophage Cholesterol Homeostasis 489