- Add 1μL of 50 mM A-769662 stock for a final concentration
of 100μM or desired AMPK activator. - Incubate in a humidified 5% CO 2 incubator at 37C for 2–18 h
(seeNote 18). - Remove the medium and wash the cells twice with 1PBS.
- Initiate the efflux of cholesterol by adding 0.5 mL of HDL
efflux medium, ApoA-I efflux medium, or strict BMDM efflux
media as a control. Importantly, AMPK activators are either
replenished or introduced at this step (seeNote 19). - Incubate cells in a humidified 5% CO 2 incubator at 37C for
4–24 h (seeNote 20). - Remove the medium from each condition into a 1.5 mL
microfuge tube. - Spin down efflux media at 4000gfor 15 min, and then
transfer supernatant into a new 1.5 mL microfuge tube. - Pipette an aliquot of each efflux medium sample into a 7 mL
scintillation vial containing 4 mL of liquid scintillation fluid. - Wash the plates twice with 1PBS.
- Lyse cells by adding 0.5 mL of 0.1 M NaOH to each well and
place on rocker at 4C overnight. - Remove an aliquot of each cell suspension sample into a 7 mL
scintillation vial containing 4 mL of liquid scintillation fluid. - Count all scintillation samples using LSC (seeNote 21).
- Calculate the percent cholesterol efflux of each sample by:
Cholesterol Efflux¼Radioactivity in medium
Radioactivity in mediumþRadioactivity in cells 100 %:- Calculate the percent-specific efflux to HDL by:
% Specific Cholesterol HDL Efflux¼HDL Efflux %Basal
Efflux % - Calculate the percent-specific efflux to ApoA-I by:
% Specific Cholesterol ApoA-I Efflux ¼ ApoA-I Efflux
%Basal Efflux %
3.5 In Vivo Reverse
Cholesterol Transport
- Plate differentiated BMDM at 5 106 cells in 100 mm plates.
- Conjugate lipid-loading medium with 5μCi/mL of^3 H-cho-
lesterol overnight at 37C. - Lipid-load the cells by adding 10 mL of lipid-loading medium
supplemented with 5μCi^3 H-cholesterol to plated BMDMs.
488 Nicholas D. LeBlond and Morgan D. Fullerton