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- [Narrator] The overall
goal of this procedure

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is to effectively isolate
and characterize macrophages

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from tissues commonly affected
by diet-induced inflammation

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such as the liver.

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- This method can help
answer key questions

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in the fields of
immunology and inflammation

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about how tissue-resident
macrophage phenotype can dictate

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the progression of chronic
inflammatory diseases.

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The main advantage of this

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is that the procedure optimizes
the isolation steps needed

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for obtaining single-cell suspensions

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derived from tissues impacted

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by obesity-mediated low-grade
chronic inflammation.

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Demonstrating the procedure
will be Joselyn Allen,

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a graduate student from my laboratory.

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- [Narrator] To isolate the liver,

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first place an ethanol-soaked
mouse in the supine position

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on a polystyrene foam dissecting board

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and secure the fore and hind paws.

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Next, use a medium point tip forceps

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to grasp the abdominal skin
near the urethra opening

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and use sharp dissecting scissors

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to create a small incision
in the raised skin.

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Insert the lower blade of the scissors

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into the incision between
the skin and the peritoneum

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and make a lateral incision
from the groin to the chin.

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Gently pull back the skin

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to expose the intact peritoneal
cavity and thoracic wall

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and make a lateral incision
through the peritoneum.

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Lift the sternum and carefully
incise the diaphragm.

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Then move the gastrointestinal
organs to the side

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and locate the subhepatic
inferior vena cava or IVC.

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Using hemostatic forceps,

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clamp the suprahepatic IVC to
maintain a localized perfusion

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and attach a syringe filled
with pre-warmed perfusion buffer

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to a 23 gauge blood
collection and infusion set.

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Gently depress the plunger
until the tubing and needle

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are filled with perfusion buffer

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and insert the needle parallel
to the subhepatic IVC.

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- Proper cannulation of the IVC

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ensures that the dissociation
buffer is locally distributed

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throughout the liver
and peripheral tissues

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optimizing the tissue digestion.

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- [Narrator] Use a four
centimeter hemostatic clamp

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to secure the needle in place

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and gently depress the plunger
to begin the perfusion.

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If the needle is appropriately positioned,

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the color will rapidly
flush from the liver

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and the lobes will become enlarged.

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Promptly cut the portal vein
to allow the perfusion buffer

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to flow freely through the liver,

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occasionally gently massaging the lobes

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between the thumb and forefinger

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to facilitate the removal of the blood.

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When only five milliliters
of perfusion buffer are left,

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replace the perfusion buffer syringe

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with a syringe filled with
pre-warmed dissociation buffer,

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continuing to gently massage the lobes

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and perfuse the liver
until it is fully digested.

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Confirm a successful digestion

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by gently pressing the blunt
edge of a pair of forceps

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into the left lateral lobe.

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An indentation should
appear on the surface

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that slowly fills once
the forceps is removed.

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Then remove the needle

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and use a short straight blade

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from a pair of dissecting scissors

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to carefully cut through
the connecting ligaments

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to excise the whole liver
and the gallbladder.

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Transfer the liver to a 10 centimeter dish

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containing 10 milliliters of ice cold DMEM

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and use toothed forceps

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to grip the severed suprahepatic IVC.

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Using medium point forceps,

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apply a rapid stroking motion to each lobe

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to release the cells from
the Glisson's Capsule

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and pass the saturated DMEM

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through a 70 micrometer cell strainer

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in a 50 milliliter conical tube on ice.

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- High yields of viable
cells can only be achieved

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by properly dissociating the liver cells

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from the Glisson's Capsule.

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- [Narrator] When all of the
cells have been collected,

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invert the tube to gently
mix the cell suspension

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and pellet the cells by centrifugation.

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Transfer the supernatant to a
new 50 milliliter conical tube

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for three more low-spin centrifugations.

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After the last centrifugation,

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transfer the supernatant
to a new 50 milliliter tube

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and centrifuge the cells one
more time at a higher speed.

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Then resuspend the
non-parenchymal cell pellet

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in FACS buffer for counting

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and dilute the liver cells to
the appropriate concentration

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according to their downstream application

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in labeled five milliliter
round bottom polystyrene tubes.

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Mice fed a high fat diet or a
high fat high cholesterol diet

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exhibit an increased infiltration

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of classically activated M1 macrophages

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in affected tissues such as the aorta.

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RON receptor expressing aortic

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CD45 positive F4/80 positive macrophages

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which demonstrate an
anti-inflammatory phenotype

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are decreased in high fat
high cholesterol diet fed mice

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while pro-inflammatory CD45
positive F4/80 positive

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CD11c positive macrophages
are found in increased numbers

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in aortas isolated from these animals.

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Further, sorted RON receptor
expressing macrophages

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derived from digested aortas
display an increased expression

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of Arginase 1 gene, a
well-established M2 macrophage marker.

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Indeed, the characterization
of liver-resident macrophages

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reveals the prevailing phenotype

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of RON receptor expressing subpopulations

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with CD11c positive pro-inflammatory
macrophage populations

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demonstrating a decreased
expression of genes

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that are strongly associated

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with the ant-inflammatory M2 phenotype.

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Similar trends are observed
in the macrophage populations

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isolated from digested
white adipose tissue

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with pro-inflammatory phenotype
macrophage populations

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exhibiting a decreased surface expression

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of the RON receptor.

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- Once mastered, this
technique can be completed

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in 20 to 30 minutes per animal
if it is performed properly.

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Following this procedure,

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other methods such as flow cytometry,

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fluorescent-activated cell
sorting, and/or quantitative PCR

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can be performed to further
characterize the phenotype

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of the isolated liver-derived
tissue-resident macrophages.

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After watching this video,

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you should have a good understanding

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of how to properly isolate
tissue-resident macrophages

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from diseased livers.

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Thanks for watching and good
luck with your experiments.

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