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In This Article

  • Overview
  • Protocol
  • Results
  • Materials

Overview

This video describes a procedure for mouse liver perfusion through the portal vein using collagenase solution prior to isolating liver cells.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation

  1. Preparation of solutions and culture media
    1. Prepare all culture media and buffer according to the Table of Materials.
  2. Preparation of instruments
    1. Set up a water bath (>10 L) at 42 °C, a peristaltic pump with variable speed, and clamps for holding flasks. Prepare curved scissors and forceps (Figure 1).
    2. On or adjacent to the water bath, set up a baking s....

Results

figure-results-58
Figure 1: Schematic representation of the perfusion suite. The flasks are held in place by clamps (not shown); the tubing containing fluid is swapped from the 1L flask to the 125 mL flask during the procedure is represented by the red dotted line. Note, that the oxygen feed does not bubble directly into the solutions in the flasks. The corks should also be notched to allow a close.......

Materials

NameCompanyCatalog NumberComments
1 L Erlenmeyer flaskFisher ScientificS63274
250 mL Erlenmeyer flaskFisher ScientificS63271
Silicone tubing Cole-Parmer96400-14This tubing runs from the flasks through the pump to the T connector and then to the 1.0 mL syringe that is connected to the catheter.
Tygon tubingFisher ScientificR3603Used as an adaptor between 96400-14 and pipettes and T connector.  This may also be used for the oxygen tubing.
T-connectorsCole-ParmerEW-06294-82
Quick dissconnectsFisher Scientific6150-0010
Pinch ClampsFisher Scientific6165-0002
Masterflex L/S Variable speed Pump; model 7553-70Cole-ParmerEW-07559-00Periplastic pump with variable speed
Pump head; model 7014-20Cole-ParmerEW-07014-20
Glass graduated 1.0 mL pipettes Fisher Scientific13-678
Curved non-serrated scissorsFine Science Tools14069-12
Dumont forcepsFine Science Tools11252-20
Curved forcepsFine Science Tools13009-12
10 mL syringeFisher Scientific03-377-23Only barrel of syringe will be needed
Sterlized spoonHome supply store
Cotton ball(s)Home supply store
Polyester sewing threadHome supply store
Masking tapeHome supply store
Thumb tacksHome supply store
Styrofoam pad 50 mL conical rack
Cookie/baking sheetHome supply store
Absorbant underpadsFisher Scientific14-206-64
19 L water bathFisher ScientificTSCOL19
BD Insyte Autogaurd Shielded IV Catheter 24 guageBecton Dickinson381412Plastic cathetar with retractable needle
Crystallizing dishes 100x50VWR 89000-290
Polystyrene petri dishesSigma AldrichP5481-500EA
50 mL conical tubesFisher Scientific12-565-270
Graduated pipettes (5 mL)Fisher Scientific170355
Graduated pipettes (25 mL)Fisher Scientific170357
EasyStrainer 100 µMGreiner bio-one542000100 µm filter
EasyStrainer 40 µMGreiner bio-one54204040 µm filter
REAGENTS
NameCompoundGrams (g/L)Millimolar (mM)
Buffer 1; pH 7.4NaCl8.3142
KCl0.56.7
HEPES2.410
BSA150.226
Buffer 2; pH 7.4NaCl3.966.74
KCl0.56.71
CaCl20.76.31
HEPES24100
BSA150.226
Phenol Red0.010.03
PBS; pH 7.4NaCl8137
KCl0.22.7
Na2HPO4-7H2O1.154.3
KH2PO40.21.4
OTHER REAGENTS
NameCompanyCatalog NumberComments
Collagenase Type IVSigma AldrichC5138
Isoflurane Abcamab144581

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