4 Notes
- Large volumes of L929-conditioned medium can be obtained
by simply scaling up the number of T175 flasks. Typical batches
of L929-conditioned medium can use upwards of 50 flasks,
each containing 50 mL of media. In this case, it is important to
decant and filter-sterilize all media into a large (sterile) bottle to
ensure a homogenous preparation. - Cell recovery from the femur and tibia is typically sufficient;
however, the humerus can also be isolated for collection of a
larger quantity of the bone marrow. - If removal of the femur from the pelvis proves difficult, it is
possible to cut the entire pelvis (a potential approach for those
who have little experience with tissue dissections). - Kim wipes are very useful at this step, as the wipes act as
sandpaper to remove the musculature off the bone. - After this spin, the bone marrow may still be visible with the
bones. If this is the case, the 0.5 mL tube containing the bones
can be placed into a fresh 1.5 mL tube (steps 7and 8 can be
repeated). - Be careful not to suck up the cellular pellet within the pipette
tip since this may sheer cells. - A final concentration of 20% L929 medium may be used if 15%
is not providing sufficient macrophage differentiation in
7–8 days. - It is also possible to plate cells in 150 mm dishes with 20 mL
media. - The described experiments take advantage of AMPK activators
(Table1), which are added post macrophage differentiation.
Simultaneous bone marrow differentiation and AMPK activa-
tion would likely affect this process. Moreover, it is important
to consider that when using AMPK-deficient mouse models,
differentiation of hematopoietic progenitors can be impaired. - This protocol describes the use of media containing 10% FBS.
Past works in other cell types and in other genetic models have
serum-starved cells prior to conducting the^3 H-acetate incor-
poration experiment; however, although there are growth fac-
tors in FBS that affect AMPK, we have seen that serum
starvation feeds back to induce AMPK activation. This compli-
cates the use of additional AMPK activators. Therefore, these
experiments are conducted in the presence of FBS. - Macrophages require higher concentrations of A-769662 as
compared to hepatocytes, hence the use of 100μM A-769662.
490 Nicholas D. LeBlond and Morgan D. Fullerton