Materials

• Mouse liver(s)
• Sterile razor blade or scissors
• Phosphate-buffered saline (PBS, e.g. Catalog # 37350)
• Collagenase/Hyaluronidase* (10X in DMEM, Catalog #07912)
• DNase I Solution* (1 mg/mL, Catalog #07900)
• RPMI 1640 Medium (Catalog #36750)
• Culture dish, non-treated (e.g. Catalog #38045)
• 70 μm cell strainer (e.g. 70 µm Reversible Strainer, Large, Catalog #27260)
• 50 mL conical tubes (e.g. Falcon® Conical Tubes, Catalog #38010)
• 15 mL conical tubes (e.g. Falcon® Conical Tubes, Catalog #38009)
• Recommended medium alternatives:
  • EasySep™ Buffer (Catalog #20144)
  • Phosphate-buffered saline containing 2% fetal bovine serum and 1 mM EDTA
  • If you are planning to perform EasySep™ cell isolation, use the medium specified in the kit-specific PIS.
• Ammonium Chloride Solution (Catalog #07850)
• OptiPrep™ density gradient medium (Catalog #07820)

Protocol

This protocol is designed for processing ONE mouse liver. Please adjust as needed:

1. Prepare the liver digestion medium by thawing and combining the following components:
   • 0.2 mL of Collagenase/Hyaluronidase** (10X in DMEM)
   • 0.3 mL of DNase I Solution**
   • 1.5 mL of RPMI 1640 Medium**
2. Allow the liver digestion medium to reach room temperature (15 - 25°C).
3. Perfuse the mouse liver(s) if needed.
   Note: To remove erythrocytes and circulating leukocytes, perfuse the liver via the left ventricle or hepatic portal vein before processing.
4. Harvest the liver tissue and place it in a non-treated culture dish or plate containing cold PBS.
   Note: Ensure the gallbladder is removed from the liver tissue.
5. Rinse the liver tissue with PBS to remove excess blood.
6. Add 1 mL** of liver digestion medium (from step 1) into a new dish or plate, then transfer the liver(s).
7. Use sterile scissors or a razor blade to mince the tissue into small pieces.
8. Transfer the minced liver tissue into a 50 mL conical tube.
9. Rinse the dish or plate with 1 mL** of liver digestion medium (from step 1) and add it to the 50 mL conical tube containing the minced liver tissue.
10. Incubate the sample at 37°C for 30 minutes on a shaking platform.
11. Place a 70 μm nylon mesh strainer over a new 50 mL conical tube and pre-wet the strainer with 5 mL of recommended medium.
12. Gently pour the digested liver sample over the strainer. Push the liver tissue through the strainer with the rubber end of a syringe plunger to obtain a single cell suspension.
    Note: Rinse the strainer with additional medium to maximize cell recovery and use new strainers if needed.
13. Centrifuge the sample at 300 x g for 10 minutes at room temperature with the brake on low. After centrifugation, carefully aspirate and discard the supernatant. If the mouse liver tissue was perfused, proceed to step 20.
14. Add 5 mL** of Ammonium Chloride Solution to the cell pellet to lyse the red blood cells.
15. Thoroughly mix the cell suspension by pipetting up and down.
16. Incubate the sample on ice for 10 minutes.
17. Top up the sample to 50 mL with the recommended medium.
18. Centrifuge at 300 x g for 10 minutes at room temperature with the brake on low. After centrifugation, carefully aspirate and discard the supernatant.
19. Perform parenchymal cell and debris removal with OptiPrep™ as follows:
    1. Prepare 10 mL** of 28% (v/v) OptiPrep™ solution by adding 2.8 mL of OptiPrep™ density gradient medium to 7.2 mL of recommended medium. Then, gently invert the tube to mix; do not shake or vortex. Store at room temperature until use.
    2. Add 10 mL** of the 28% (v/v) OptiPrep™ solution to the cell pellet and gently pipette up and down.
    3. Carefully transfer the sample to a 15 mL conical tube (or 50 mL when processing multiple livers) and avoid generating foam and/or bubbles.
    4. Carefully overlay 4 mL** of cold recommended medium onto the cell suspension by slowly dispensing it down the wall of the tube without disturbing the interface.
    5. Centrifuge at 1300 x g for 15 minutes at room temperature with no brake.
    6. Using a Pasteur or serological pipette, carefully collect the interface containing non-parenchymal cells and transfer to a new conical tube. See Figure 1 below for a schematic representation.
    7. Top up the new tube to 10 mL** with recommended medium and centrifuge at 300 x g for 10 minutes at room temperature with the brake on low.
    8. Carefully aspirate and discard the supernatant.
20. The cells are now ready for downstream applications. For cell separation, determine the total nucleated cell (TNC) number. If using the EasySep™ Mouse Liver F4/80 Positive Selection Kit, resuspend at 5 x 10⁷ cells/mL in EasySep™ Buffer (Catalog #20144) or PBS with 2% FBS and 1 mM EDTA.
**Note: Volume given is for processing one liver. Adjust as needed to process multiple livers.