- [Narrator] The overall goal of this procedure
is to effectively isolate and characterize macrophages
from tissues commonly affected by diet-induced inflammation
such as the liver.
- This method can help answer key questions
in the fields of immunology and inflammation
about how tissue-resident macrophage phenotype can dictate
the progression of chronic inflammatory diseases.
The main advantage of this
is that the procedure optimizes the isolation steps needed
for obtaining single-cell suspensions
derived from tissues impacted
by obesity-mediated low-grade chronic inflammation.
Demonstrating the procedure will be Joselyn Allen,
a graduate student from my laboratory.
- [Narrator] To isolate the liver,
first place an ethanol-soaked mouse in the supine position
on a polystyrene foam dissecting board
and secure the fore and hind paws.
Next, use a medium point tip forceps
to grasp the abdominal skin near the urethra opening
and use sharp dissecting scissors
to create a small incision in the raised skin.
Insert the lower blade of the scissors
into the incision between the skin and the peritoneum
and make a lateral incision from the groin to the chin.
Gently pull back the skin
to expose the intact peritoneal cavity and thoracic wall
and make a lateral incision through the peritoneum.
Lift the sternum and carefully incise the diaphragm.
Then move the gastrointestinal organs to the side
and locate the subhepatic inferior vena cava or IVC.
Using hemostatic forceps,
clamp the suprahepatic IVC to maintain a localized perfusion
and attach a syringe filled with pre-warmed perfusion buffer
to a 23 gauge blood collection and infusion set.
Gently depress the plunger until the tubing and needle
are filled with perfusion buffer
and insert the needle parallel to the subhepatic IVC.
- Proper cannulation of the IVC
ensures that the dissociation buffer is locally distributed
throughout the liver and peripheral tissues
optimizing the tissue digestion.
- [Narrator] Use a four centimeter hemostatic clamp
to secure the needle in place
and gently depress the plunger to begin the perfusion.
If the needle is appropriately positioned,
the color will rapidly flush from the liver
and the lobes will become enlarged.
Promptly cut the portal vein to allow the perfusion buffer
to flow freely through the liver,
occasionally gently massaging the lobes
between the thumb and forefinger
to facilitate the removal of the blood.
When only five milliliters of perfusion buffer are left,
replace the perfusion buffer syringe
with a syringe filled with pre-warmed dissociation buffer,
continuing to gently massage the lobes
and perfuse the liver until it is fully digested.
Confirm a successful digestion
by gently pressing the blunt edge of a pair of forceps
into the left lateral lobe.
An indentation should appear on the surface
that slowly fills once the forceps is removed.
Then remove the needle
and use a short straight blade
from a pair of dissecting scissors
to carefully cut through the connecting ligaments
to excise the whole liver and the gallbladder.
Transfer the liver to a 10 centimeter dish
containing 10 milliliters of ice cold DMEM
and use toothed forceps
to grip the severed suprahepatic IVC.
Using medium point forceps,
apply a rapid stroking motion to each lobe
to release the cells from the Glisson's Capsule
and pass the saturated DMEM
through a 70 micrometer cell strainer
in a 50 milliliter conical tube on ice.
- High yields of viable cells can only be achieved
by properly dissociating the liver cells
from the Glisson's Capsule.
- [Narrator] When all of the cells have been collected,
invert the tube to gently mix the cell suspension
and pellet the cells by centrifugation.
Transfer the supernatant to a new 50 milliliter conical tube
for three more low-spin centrifugations.
After the last centrifugation,
transfer the supernatant to a new 50 milliliter tube
and centrifuge the cells one more time at a higher speed.
Then resuspend the non-parenchymal cell pellet
in FACS buffer for counting
and dilute the liver cells to the appropriate concentration
according to their downstream application
in labeled five milliliter round bottom polystyrene tubes.
Mice fed a high fat diet or a high fat high cholesterol diet
exhibit an increased infiltration
of classically activated M1 macrophages
in affected tissues such as the aorta.
RON receptor expressing aortic
CD45 positive F4/80 positive macrophages
which demonstrate an anti-inflammatory phenotype
are decreased in high fat high cholesterol diet fed mice
while pro-inflammatory CD45 positive F4/80 positive
CD11c positive macrophages are found in increased numbers
in aortas isolated from these animals.
Further, sorted RON receptor expressing macrophages
derived from digested aortas display an increased expression
of Arginase 1 gene, a well-established M2 macrophage marker.
Indeed, the characterization of liver-resident macrophages
reveals the prevailing phenotype
of RON receptor expressing subpopulations
with CD11c positive pro-inflammatory macrophage populations
demonstrating a decreased expression of genes
that are strongly associated
with the ant-inflammatory M2 phenotype.
Similar trends are observed in the macrophage populations
isolated from digested white adipose tissue
with pro-inflammatory phenotype macrophage populations
exhibiting a decreased surface expression
of the RON receptor.
- Once mastered, this technique can be completed
in 20 to 30 minutes per animal if it is performed properly.
Following this procedure,
other methods such as flow cytometry,
fluorescent-activated cell sorting, and/or quantitative PCR
can be performed to further characterize the phenotype
of the isolated liver-derived tissue-resident macrophages.
After watching this video,
you should have a good understanding
of how to properly isolate tissue-resident macrophages
from diseased livers.
Thanks for watching and good luck with your experiments.
(low bass techno music)