Mouse Lung Digestion to Single-Cell Suspension

Dillon Corvino

Enzymatic digestion of mouse lung to generate viable single-cell suspensions for flow cytometry, sorting, and single-cell omics workflows.
Published

September 18, 2026

Protocol ID: MUS-LNG-DIG
Version: v1.1
Author: Dillon Corvino

Purpose

This protocol describes the enzymatic and mechanical dissociation of mouse lung tissue to obtain single-cell suspensions suitable for flow cytometry, FACS sorting, and single-cell sequencing. Lung tissue is fibrous and elastic; therefore, digestion requires both collagenase and DNase together with thorough mechanical disruption.

Critical notes (read before starting)

  • Keep all buffers and tubes cold except during digestion at 37 °C.
  • Use fresh, pre-warmed digestion buffer with DNase added on the day of use.
  • Lung tissue contains trapped blood; incomplete perfusion increases RBC contamination.
  • Do not over-digest: limit digestion to 30–35 minutes, as prolonged incubation reduces viability.
  • After digestion, perform all downstream steps on ice.
  • Use 70 μm strainers and do not reuse between lungs.

Approximate timing

  • Dissection: 5 min
  • Mince + pre-digestion handling: 5 min
  • Enzymatic digestion: 30–35 min
  • Mechanical processing and washes: 20–25 min

Total hands-on time: ~60 minutes per lung (less when batching).

Procedure

STEP 1 – Lung collection

  1. Euthanise the mouse using an approved method.
  2. Open the thoracic cavity to expose the lungs.
  3. (Optional but recommended) Perfuse via the right ventricle with cold PBS until lungs visibly blanch, reducing RBC content.
  4. Excise the entire lung block and place it in a 10 cm petri dish containing cold PBS or FACS/MACS Buffer.
Tip

Perfusion markedly improves final leukocyte purity and reduces RBC lysis requirements.

STEP 2 – Tissue mincing

  1. Blot the lungs briefly on sterile tissue to remove excess fluid.
  2. Transfer to a fresh 10 cm dish and mince thoroughly using scissors or scalpels until small fragments are obtained.
  3. Transfer the minced lung into a 50 mL Falcon tube containing 5 mL Lung Digestion Buffer (see buffer protocol BUF-LNG-DIG-v1.0).

STEP 3 – Enzymatic digestion

  1. Place tubes on a 37 °C benchtop shaker at 250 rpm.
  2. Incubate for 30–35 minutes.
Warning

Over-digestion can decrease viability and negatively impact epithelial and myeloid populations.

STEP 4 – Mechanical dissociation

  1. After digestion, add FACS/MACS Buffer to bring total volume to 25 mL.
  2. Pour the suspension onto a 70 μm cell strainer placed on a 10 cm dish.
  3. Mechanically mash the digested lung through the mesh using the back of a 5 mL syringe plunger.
  4. Rinse the strainer with cold FACS/MACS Buffer until no visible tissue remains.
  5. Transfer the filtered cell suspension back into a 50 mL tube.
  6. Top up to 50 mL with cold FACS/MACS Buffer.
  7. Centrifuge at 400 g for 5 minutes at 4 °C.
  8. Carefully discard the supernatant.

STEP 5 – RBC removal (if required)

Lung digests vary in RBC content. If erythrocytes are visible:

  1. Add 1 mL 1× RBC Lysis Buffer (e.g. HybriMax) per lung.
  2. Mix gently and incubate for 3–5 minutes at room temperature.
  3. Immediately top up to 50 mL with cold FACS/MACS Buffer.
  4. Centrifuge at 400 g for 5 minutes at 4 °C.
  5. Discard supernatant.
Note

If the lung was perfused effectively, this step may not be needed.

STEP 6 – Final wash and resuspension

  1. Loosen pellet by gentle flicking or brief vortexing.
  2. Resuspend in 5 mL FACS/MACS Buffer (or other desired volume).
  3. Filter again through a fresh 70 μm strainer if clumping is observed.
  4. Keep the suspension on ice until staining, sorting, or single-cell workflows.

Buffers Required

Full composition, preparation, storage, and safety information for each buffer lives in its own linked buffer document — only a concise per-buffer summary is reproduced here, generated automatically from that document.

FACS Buffer

Component Final concentration / amount
PBS 1x to 500 mL
FCS 10 mL (2% v/v)
  • Final volume: 500 mL
  • Preparation note: Mix gently by inversion.
  • Sterilisation / filtering: 0.22 um filter if sterility required
  • Storage: 4C, 4-6 weeks
  • Caveat: No EDTA - EDTA is excluded from FACS buffer to avoid interference with staining reagents and functional assays. Use MACS Buffer (BUF-MACS-BUF) instead if EDTA is needed.

Full composition, preparation steps, storage, and safety information: BUF-FACS-BUF.

MACS Buffer

Component Final concentration / amount
PBS 1x to 500 mL
FCS 10 mL (2% v/v)
EDTA (0.5 M) 2 mL (2 mM final)
  • Final volume: 500 mL
  • Preparation note: Mix gently by inversion.
  • Sterilisation / filtering: 0.22 um filter if sterility required
  • Storage: 4C, 4-6 weeks
  • Caveat: Contains EDTA - do not use for liver perfusion or any step upstream of collagenase digestion (EDTA chelates the Ca2+/Zn2+ cofactors collagenases require). Use FACS Buffer (BUF-FACS-BUF) instead.

Full composition, preparation steps, storage, and safety information: BUF-MACS-BUF.

RBC Lysis Buffer (HybriMax)

Component Final concentration / amount
RBC Lysis Buffer (HybriMax), 1x ready-to-use as supplied
  • Final volume: 0.5 mL per sample (as used in MUS-LIV-DIG)
  • Preparation note: If supplied as concentrate, dilute to 1x per manufacturer instructions before use.
  • Storage: 2-8C per manufacturer instructions; do not freeze unless explicitly supported
  • Caveat: Used at 0.5 mL of 1x buffer per sample with a 7-minute room-temperature incubation in MUS-LIV-DIG - adapt incubation time if switching supplier.

Full composition, preparation steps, storage, and safety information: BUF-RBC-HYB.

ACK RBC Lysis Buffer

Component Final concentration / amount
Ammonium chloride (NH4Cl) 8.26 g/L
Potassium bicarbonate (KHCO3) 1.00 g/L
EDTA 0.037 g/L (37 mg/L)
  • Final volume: 1 L
  • Preparation note: Dissolve in sterile distilled water, then adjust to pH 7.2 with dilute HCl before sterile filtration.
  • Sterilisation / filtering: 0.22 um filter required
  • Storage: 4C; stable up to 3 months when sterile; do not freeze.

Full composition, preparation steps, storage, and safety information: BUF-ACK-LYSIS.

Materials

Reagents

Reagent Supplier / Cat# Notes
Lung Digestion Buffer In-house Pre-warmed to 37 °C
FACS/MACS Buffer In-house BUF-FACS-BUF / BUF-MACS-BUF; cold
RBC Lysis Buffer (1×) [TBD] Use only if needed
PBS (1×) Any For perfusion and rinsing

Disposables

Item Notes
50 mL Falcon tubes 1 per lung
10 cm petri dishes For mincing & filtering
70 μm strainers Do not reuse
5 mL syringes (no needle) For mechanical dissociation
Scissors, scalpels For mincing
Transfer pipettes For washing & transfers

Equipment

Equipment Notes
Benchtop shaker/incubator 37 °C, 250 rpm
Benchtop centrifuge 400 g at 4 °C
Biosafety cabinet (Class II) Recommended
Timer For digestion and lysis steps

Troubleshooting

Issue Possible cause Suggested solution
Persistent clumps Incomplete digestion Increase mechanical dissociation; do not over-digest
High RBC contamination Lungs not perfused Perform RBC lysis or perfuse before collection
Low viability Over-digestion or RT handling Limit digestion to 30–35 min; keep washes cold
Excess debris Harsh mechanical disruption Use gentle, repetitive plunger strokes

Safety (brief)

  • Treat mouse tissues as potentially infectious and process according to institutional S1/S2 rules.
  • Enzyme mixtures (Collagenase, DNase) may cause irritation; wear gloves and eye protection.
  • Dispose of sharps, tissues, and enzymatic waste according to facility guidelines.

Version history

Version Date Author Change summary
v1.0 2025-11-20 Dillon Corvino Initial upgraded version for lung digestion
v1.1 2026-09-18 Dillon Corvino Replaced # Buffers used (bare library(tidyverse), leaked R package-startup warnings into the rendered document) with an auto-generated # Buffers Required section, kept in its correct position before Materials; Materials table FACS/MACS Buffer row now lists both BUF-FACS-BUF and BUF-MACS-BUF (registry split into separate EDTA-free/EDTA-containing buffers; prose wording left as “FACS/MACS Buffer” since the distinction does not matter to this procedure).