- Longer time points could potentially skew interpretation given
the metabolism of lipids into which^3 H-acetate has been
incorporated. This may not reflect only synthesis. - At this point, the chloroform (lipid-containing) phase can be
evaporated to dryness under a stream of nitrogen gas for TLC
separation or saponified with KOH to extract^3 H-containing
fatty acid and sterols, as described in other chapters of this
issue. - To calculate the nmol incorporation of^3 H-acetate into choles-
terol, use the molar amount of sodium acetate and the radio-
activity in the initial media to determine specific activity. - If cell debris is present in tubes, this can be pelleted by cen-
trifuging at 12,000gfor 5 min and the supernatant trans-
ferred into a fresh 1.5 mL tube. - It is important to note that the uptake of Dil-acLDL can be
assessed under any permutation of lipid-loaded and AMPK
activation treatments. The protocol detailed above assesses
Dil-acLDL uptake in basal BMDM. Past work has demon-
strated that there is no difference between wild-type and
AMPKβ1-deficient macrophages in the presence or absence
of A-769662 treatment. - Each respective plate should be done in triplicate independent
of how many technical replicates within treatment groups. One
plate is to be used to assess HDL-mediated efflux, one for
ApoA-І-mediated efflux, and basal efflux (used to calculate
the percent-specific efflux). - During the equilibration treatments, AMPK activators may be
added to “prime” the system prior to the efflux phase of the
experiment. Shorter equilibration times may be used ranging
from 2 to 18 h. - It is important to consider that AMPK activator treatment and
AMPK deficiency can and do alter total cellular cholesterol
pools. For example, if A-769662 is added to macrophages
during the initial lipid-loading phase, there is a significant
reduction in total cholesterol content, which confounds the
assessment of^3 H-cholesterol. For this reason, it is critical to
also measure total cellular cholesterol pools when introducing
new AMPK genetic models or pharmacological activators/
inhibitors. To this point, timing of AMPK activator treatment
can also be tailored to suit investigations into transcriptional
vs. acute posttranslational effects by modulating overall
treatment time. - Kinetics of cholesterol efflux in response to various AMPK
activators can be observed by including numerous time points. - Scintillation vials can be left to equilibrate overnight and
should be counted after at least 24 h.
Assessing Macrophage Cholesterol Homeostasis 491