Mouse Liver Digestion Buffer

Dillon Corvino

Collagenase and DNase I digestion buffer for enzymatic dissociation of mouse liver tissue. Supports Collagenase D or Collagenase IV in a DMEM, PBS, or HBSS base.
Published

September 18, 2026

Protocol ID: BUF-LIV-DIG Version: v1.1 Author: Dillon Corvino Date: September 18, 2026

Purpose

This buffer is used for enzymatic digestion of mouse liver tissue to generate viable single-cell suspensions. It contains a collagenase and DNase I at working concentrations suitable for dissociating liver parenchyma while preserving lymphocyte viability.

This single buffer replaces the former separate Collagenase D and Collagenase IV documents (BUF-LIV-DIG-COL-D, BUF-LIV-DIG-COL-IV, both retired at v2.0 of MUS-LIV-DIG).

Linked protocols

  • MUS-LIV-DIG

Choice of collagenase and base medium

Two collagenases are in routine use. They are not interchangeable at the same concentration — Collagenase D is used at twice the concentration of Collagenase IV.

Enzyme Working concentration Notes
Collagenase D 1 mg/mL Roche 11088858001; supplied as lyophilised powder
Collagenase IV 0.5 mg/mL Half the concentration used for Collagenase D

DNase I is 50 ug/mL for both.

The base medium may be DMEM (incomplete), PBS 1x, or HBSS 1x — these are interchangeable for this application. Use whichever is on hand; HBSS and PBS avoid the phenol red background in DMEM if that matters downstream.

Important

Do not use an EDTA-containing buffer (e.g. MACS Buffer) as the base medium, and do not perfuse the liver with one. Collagenases are Ca2+/Zn2+-dependent metalloproteases and EDTA chelates these cofactors, inhibiting digestion.

Working buffer composition

Enzyme Final volume (mL) Base medium Collagenase (100 mg/mL stock) DNase I (10 mg/mL stock)
Collagenase D 1 to 1 mL 10 uL 5 uL
Collagenase D 5 to 5 mL 50 uL 25 uL
Collagenase D 10 to 10 mL 100 uL 50 uL
Collagenase D 25 to 25 mL 250 uL 125 uL
Collagenase D 50 to 50 mL 500 uL 250 uL
Collagenase IV 1 to 1 mL 5 uL 5 uL
Collagenase IV 5 to 5 mL 25 uL 25 uL
Collagenase IV 10 to 10 mL 50 uL 50 uL
Collagenase IV 25 to 25 mL 125 uL 125 uL
Collagenase IV 50 to 50 mL 250 uL 250 uL

5 mL of working buffer is required per liver.

Preparation

A. Preparation of stock (if applicable)

Reagent Stock concentration Storage
Collagenase D or IV 100 mg/mL in sterile PBS Aliquot at -20 °C
DNase I 10 mg/mL Aliquot at -20 °C; avoid repeated freeze-thaw

Reconstitution of Collagenase powder. Collagenase D is supplied as a lyophilised powder and must be reconstituted before use.

  1. Add 1 mL sterile PBS 1x directly to a vial containing 100 mg Collagenase powder.
  2. Swirl gently until fully dissolved; do not vortex.
  3. This yields a 100 mg/mL stock.
  4. Aliquot into single-use volumes and store at -20 °C, labelled with enzyme, concentration, date, and initials.

B. Preparation of working buffer

  1. Thaw collagenase and DNase I aliquots on ice.
  2. Add the base medium (DMEM, PBS, or HBSS) to a sterile tube.
  3. Add collagenase and DNase I per the table above.
  4. Mix gently by inversion; do not vortex.
  5. Pre-warm to 37 °C immediately before use.
Note

Collagenase-only buffer (without DNase) may be prepared in advance and frozen in aliquots. Add DNase I fresh on the day of use.

Storage and stability

  • Keep freshly prepared working buffer on ice until pre-warming for use.
  • Do not freeze buffer after DNase I has been added.
  • Discard unused working buffer at the end of the day.

Reagent details

Component Supplier Cat. # Notes
Collagenase D Roche, Basel, Switzerland 11088858001 Lyophilised powder
Collagenase IV [TBD] [TBD]
DNase I Sigma-Aldrich, Merck DN25-100MG
DMEM (incomplete) [TBD] [TBD]
HBSS 1x PAN-Biotech, Aidenbach, Germany P04-34500
DPBS, no Ca / no Mg Gibco, ThermoFisher 14190-094

Safety (brief)

  • Collagenase and DNase I are enzymes of biological origin — handle with standard laboratory PPE and avoid aerosolisation.
  • Mouse liver tissue is a biological sample — handle and dispose of according to institutional biosafety rules.
  • Dispose of enzyme-containing waste per facility biological/chemical waste procedures.

Version history

Version Date Author Change summary
v1.0 2026-08-05 Dillon Corvino Created by merging BUF-LIV-DIG-COL-D and BUF-LIV-DIG-COL-IV. DNase I raised to 50 ug/mL for both enzymes (was 10 ug/mL for Col D, 25 ug/mL for Col IV). Base medium stated as DMEM, PBS, or HBSS.
v1.1 2026-09-18 Dillon Corvino Migrated to shared summary:/helper architecture (buffer/protocol redesign); fixed leaked R package-startup warnings in “Linked protocols”; added # Safety (brief); renamed # Materials to # Reagent details; restructured Preparation into A/B sub-headings.