How to Manually Dissociate Mouse Liver Tissue into a Single-Cell Suspension
This protocol describes a simple and reliable method for manually processing mouse liver tissue into a single-cell suspension. The workflow combines mechanical disruption (i.e. tissue mincing) with enzymatic digestion using Collagenase/Hyaluronidase and DNase I to release viable cells and reduce cell clumping. After filtration, red blood cell lysis, and density gradient separation, the resulting cell suspension is compatible with downstream applications, including EasySep™ cell isolation, which enables simple, column-free enrichment of target cell populations such as F4/80⁺ cells. Depending on the target cell type and downstream application (e.g. magnetic cell isolation, flow cytometry, cell culture, etc.), modifications such as buffer selection may be required. For recommended sample preparation guidelines, refer to the EasySep™ kit-specific Product Information Sheet (PIS).
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 according to your cell isolation kit's Product Information Sheet (PIS). Recommended options include:
- EasySep™ Buffer (Catalog #20144)
- Phosphate-buffered saline containing 2% fetal bovine serum and 1 mM EDTA
- 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:
Before You Begin
- Processing multiple livers? Adjust reagent volumes accordingly. Do not exceed two livers per 50 mL conical tube.
- Prefer 15 mL conical tubes? Use at step 20 and process one liver per tube (tip: 50 mL tubes are usually easier to handle).
- Performing EasySep™ cell isolation? Process at least two mouse livers for optimal results.
- 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
- Allow the liver digestion medium to reach room temperature (15 - 25°C).
- Perfuse the mouse liver(s) if needed.
- Harvest the liver tissue and place it in a non-treated culture dish or plate containing cold PBS.
- Rinse the liver tissue with PBS to remove excess blood.
- Add 1 mL** of liver digestion medium (from step 1) into a new dish or plate, then transfer the liver(s).
- Use sterile scissors or a razor blade to mince the tissue into small pieces.
- Transfer the minced liver tissue into a 50 mL conical tube.
- 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.
- Incubate the sample at 37°C for 30 minutes on a shaking platform.
- 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.
- Gently pour the digested liver sample over the strainer. Push the liver tissue through with the rubber end of a syringe plunger to obtain a single cell suspension.
- Rinse the strainer with additional medium to maximize cell recovery. Use new strainers if needed.
- 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.
- Add 5 mL** of Ammonium Chloride Solution to the cell pellet to lyse the red blood cells.
- Thoroughly mix the cell suspension by pipetting up and down.
- Incubate the sample on ice for 10 minutes.
- Top up the sample to 50 mL with the recommended medium.
- Centrifuge at 300 x g for 10 minutes at room temperature with the brake on low. After centrifugation, carefully aspirate and discard the supernatant.
- Perform parenchymal cell and debris removal with OptiPrep™ as follows:
- 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.
- Add 10 mL** of the 28% (v/v) OptiPrep™ solution to the cell pellet and gently pipette up and down.
- Carefully transfer the sample to a conical tube and avoid generating foam and/or bubbles.
- 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.
- Centrifuge at 1300 x g for 15 minutes at room temperature with no brake.
- 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.
- Top up the new tube to 10 mL** with recommended medium and centrifuge at 300 x g for 10 minutes.
- Carefully aspirate and discard the supernatant.
- 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.
Figure 1. OptiPrep™ Separation of Mouse Liver Non-Parenchymal Cells.
Schematic representation of OptiPrep™ density gradient separation following centrifugation. The four layers generated, from bottom to top, are as follows: (1) pellet containing parenchymal cells and debris (red), (2) 28% (v/v) OptiPrep™ layer (white), (3) interface depicting enriched non-parenchymal cells to be carefully collected (orange), and (4) overlaid recommended medium (gray).
Did you know ƽ Technologies offers a broad portfolio of EasySep™ Mouse Cell Isolation Kits? Discover simplified, column-free magnetic cell isolation for a wide range of mouse cell populations.
If you have any questions, contact us at techsupport@stemcell.com.
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