Mouse Liver Digestion to Single-Cell Suspension

Dillon Corvino

Isolation of viable mouse liver single-cell suspensions for downstream flow cytometry, sorting, and single-cell applications.
Published

September 18, 2026

Protocol ID: MUS-LIV-DIG
Version: v2.1
Author: Dillon Corvino

Purpose

This protocol describes the preparation of single-cell suspensions from mouse liver using perfusion, enzymatic digestion, mechanical dissociation, density centrifugation, and RBC lysis. The resulting cell suspension is suitable for downstream applications such as flow cytometry, FACS sorting, and single-cell omics workflows (with appropriate QC).

Workflow summary

Perfuse (ice-cold PBS) -> gentleMACS digestion -> 70 um filtration with syringe plunger -> wash spins -> Percoll -> RBC lysis

Critical notes (read before starting)

  • Perfuse with ice-cold PBS or FACS Buffer only — never MACS Buffer. See the callout in STEP 2.
  • Spin times depend on tube format: 50 mL tubes 400 g for 7 min, 15 mL tubes 400 g for 5 min. See the callout in STEP 5.
  • Percoll enrichment is performed in a 50 mL tube, not a 15 mL tube. See the callout in STEP 6.
  • Keep all post-digestion washes cold (4 °C) where possible to preserve viability.
  • Process one liver per 70 µm strainer; do not reuse strainers between livers.
  • Liver cell pellets are often loose; be careful when pouring supernatants.
  • Do not over-digest; limit digestion to the programmed gentleMACS time, or 30 minutes at 37 °C with agitation if dissociating manually.
  • Percoll must be at room temperature and properly diluted to 37% of isotonic stock (= 33% final Percoll) immediately before use.
  • Ensure RBC lysis is time-limited (2 minutes with ACK) to avoid damaging leukocytes.

Approximate timing

  • Preparation and setup: 10–15 min
  • Mouse euthanasia, perfusion, and liver dissection: 5–10 min per mouse
  • gentleMACS dissociation and enzymatic digestion: 25–30 min
  • 70 µm filtration: 5 min per liver
  • Two wash spins (400 g, 7 min each in 50 mL tubes): 20–25 min
  • Percoll centrifugation and leukocyte enrichment: 20–25 min
  • RBC lysis, quench, and final resuspension: 15–20 min

Total approximate time: ~2–2.5 hours for 1–4 livers processed in parallel
(Effective hands-on time per liver is ~60–90 minutes, depending on batching and operator experience.)

Prep checklist

Everything below is per mouse. Multiply by the number of animals, and add margin on the buffers so the last sample is not short.

Reagents

Reagent Per mouse Where it is used
PBS or FACS Buffer, ice-cold (BUF-FACS-BUF) 10 mL STEP 2 — perfusion (5–10 mL, until the liver blanches). Perfusion only; never MACS Buffer
Liver digestion buffer (BUF-LIV-DIG) 5 mL STEP 4 — pre-warmed to 37 °C in the C-tube
37% Percoll (BUF-PERC-37) 25 mL STEP 6 — from isotonic stock: 9.25 mL stock + 15.75 mL PBS
ACK lysis buffer (BUF-ACK-LYSIS) 1 mL STEP 7
MACS Buffer (BUF-MACS-BUF) ~110 mL Collection 5 mL; STEP 5 washes 35 + 40 mL; STEP 7 quench 9 mL; STEP 8 final 5 mL (94 mL used, allowance includes margin)

Plasticware and consumables

Item Per mouse Note
gentleMACS C-tube 1 Option A only
70 um cell strainer 1 Strictly one per liver — never reuse between livers
50 mL conical tube 3 Collection (STEP 3), filtration + washes (STEP 5), Percoll (STEP 6). Add 1 if using the optional 50 g hepatocyte spin
15 mL conical tube 1 Post-lysis wash (STEP 7)
5 mL syringe 1 Plunger only — used to press tissue through the strainer (STEP 5)
Syringe, 5–10 mL 1 Perfusion; may be shared within a treatment group
Needle, 25–27 G 1 Perfusion
Transfer pipette 2 Pellet resuspension (STEP 6), ACK resuspension (STEP 7)
Filter paper 1 piece Blotting the liver before weighing
Note

Collection at STEP 3 may instead use 1 well of a 6-well plate with 3–5 mL ice-cold buffer, in which case only 2 50 mL tubes are needed per mouse.

Shared, not per mouse: gentleMACS + warming sleeves, 37 °C water bath, refrigerated centrifuge with adjustable brake and acceleration, 5–10 mL stripettes, paper towels, ice, timers, dissection scissors and forceps, sharps container, analytical balance.

Note

Full recipes, stock preparation, and storage for each buffer are no longer reproduced here — see the auto-generated # Buffers Required section after the Procedure below, or open the linked buffer document directly (BUF-LIV-DIG, BUF-PERC-37, BUF-FACS-BUF, BUF-MACS-BUF, BUF-ACK-LYSIS).

Procedure

STEP 1 – Preparation and setup

  1. Pre-cool collection solutions
    • Pre-cool PBS on ice for liver perfusion.
    • Pre-cool MACS Buffer on ice for tissue collection and all subsequent washes.
  2. Label collection vessels (choose one option per experiment)
    • Option A: 6-well plate collection
      • Label one well per mouse in a 6-well plate.
      • Add 3–5 mL ice-cold PBS or MACS Buffer per well.
    • Option B: 50 mL tube collection
      • Label one 50 mL conical tube per liver.
      • Add 5 mL ice-cold PBS or MACS Buffer per tube.
  3. Label dissociation and processing tubes
    • Label gentleMACS C-tubes (or equivalent) for each liver.
    • Label 50 mL tubes for filtration, washing, and Percoll enrichment (one per liver).
    • Label 15 mL tubes for the post-lysis wash (one per liver).
  4. Prepare buffers
    • Liver digestion buffer — BUF-LIV-DIG; 5 mL per liver.
    • 37% Percoll working solution — BUF-PERC-37; 25 mL per liver (9.25 mL isotonic stock + 15.75 mL PBS). Add margin when preparing for several livers so the last tube is not short.
    • ACK lysis buffer — 1 mL per liver.
    • MACS Buffer for washes and final resuspension — allow ~110 mL per liver.
  5. Warm digestion components
    • Pre-warm a water bath to 37 °C.
    • Place an aliquot of liver digestion buffer in the water bath to equilibrate.
    • If digestion buffer will be prepared immediately before use, pre-warm the base medium (DMEM, PBS, or HBSS) instead.
  6. Equipment setup
    • Pre-cool a benchtop centrifuge to 4 °C.
    • Ensure access to a gentleMACS instrument with an appropriate liver dissociation program.
  7. Items to take to the animal facility
    • Ice bucket with ice-cold PBS for perfusion.
    • 6-well plates and/or pre-filled 50 mL tubes (as selected above).
    • Sterile scissors and forceps.
    • 5–10 mL syringes (one per mouse or per treatment group).
    • Needles for liver perfusion (25–27 G recommended).
    • Absorbent pads and a waste container for perfusion runoff.

STEP 2 – Liver harvest and perfusion

  1. Euthanise the mouse according to institutional and ethical guidelines (e.g. CO₂ asphyxiation or other approved method).
  2. Place the mouse in a supine position and perform a midline incision to open the abdominal cavity.
  3. Expose the liver and major vessels (Inferior vena cava and portal vein).
  4. Load a 5 mL syringe with ice-cold PBS and attach a 25–27 G needle.
  5. Perfuse the liver via one of the following routes:
    • Inferior vena cava
    • Portal vein
  6. Slowly perfuse with ~5–10 mL ice-cold PBS per mouse until the liver visibly blanches.
Important

Perfuse with PBS or FACS Buffer only — do not perfuse with MACS Buffer.
MACS Buffer contains 2 mM EDTA. Anecdotal evidence suggests residual EDTA carried into the tissue inhibits downstream collagenase digestion (collagenases are Ca2+/Zn2+-dependent metalloproteases and EDTA chelates these cofactors).
MACS Buffer is fine for all subsequent steps — collection, washes, Percoll resuspension, quenching, and final resuspension.

Note

Efficient perfusion reduces blood content and improves hepatocyte removal, density separation, and downstream staining quality.

Warning

Handle needles and sharps carefully and dispose of them immediately into an approved sharps container after perfusion.

  1. Excise the liver carefully, removing the gallbladder.
  2. Place the liver briefly on filter paper to blot excess buffer.

STEP 3 – Liver collection, weighing, and mincing

  1. Transfer the perfused liver to a pre-labelled collection vessel (on ice).
  2. (Recommended) Weigh the liver:
    • Briefly blot on filter paper to remove excess liquid.
    • Record the liver weight.

STEP 4 – enzymatic and mechanical dissociation

Option A - GentleMACS C-tube

  1. Add 5 mL pre-warmed liver digestion buffer (with DNase) to a labelled gentleMACS C-tube.
  2. Transfer the liver into the gentleMACS C-tube.
  3. Secure the C-tube onto the gentleMACS instrument and place the warming sleeve over the tube.
  4. Run the m_liver_02_02 program to mechanically dissociate the tissue.
  5. After the program finishes, briefly inspect the tube to ensure the tissue has been mechanically disrupted into small fragments and is fully submerged in digestion buffer.
  6. Run the HH17 program (~17 min) to allow warmed enzymatic digestion of the tissue.
Warning

Confirm that each C-tube is correctly seated and rotating before leaving the instrument.
If a tube is not spinning, stop the run, reseat the tube, and restart the program.

Important

Do not leave livers in digestion buffer beyond the programmed digestion time.
Over-digestion can strip surface epitopes and negatively impact downstream phenotyping.
Note: the gentleMACS instrument does not beep when the program finishes—set a timer before starting.

Proceed directly to STEP 5 – Filtration.

Option B – Manual mechanical dissociation (fallback)

Use this route if the gentleMACS is unavailable.

  1. Add 5 mL pre-warmed liver digestion buffer (with DNAse) to a labelled 50 mL tube.
  2. Transfer the liver into the 50 mL tube.
  3. Using sterile scissors, mince the liver directly in the tube until fragments are approximately 2–3 mm in size.
  4. Ensure all tissue pieces are fully submerged in digestion buffer and not adhered to the tube walls.
  5. Place the tube horizontally on a 37°C shaking incubator (250 rpm) and incubate for 30 minutes.
Important

Ensure tubes are tightly closed and positioned securely on the shaker.
Set a timer — prolonged digestion can reduce viability and affect surface marker integrity.

Proceed directly to STEP 5 – Filtration.

STEP 5 – Filtration and washing

Applies to both Option A and Option B.

  1. Seat a 70 µm cell strainer on a labelled 50 mL conical tube.
  2. Pour the digested liver suspension through the strainer.
  3. Using the back of a 5 mL syringe plunger, gently press the remaining tissue through the strainer until no visible fragments remain.
  4. Rinse the strainer with ice-cold MACS Buffer while continuing gentle mechanical disruption.
  5. Bring the total volume to 40 mL with ice-cold MACS Buffer.
Note

Filtration is performed directly into the 50 mL tube — no petri dish, and no dab/swirl recovery steps. One strainer per liver; do not reuse.

  1. Centrifuge at 400 g for 7 minutes at 4 °C.
Important

Spin time depends on tube format. The longer path length in a 50 mL tube requires a longer spin to pellet equivalently:

Tube Speed Time
50 mL 400 g 7 min
15 mL 400 g 5 min

This applies to every 400 g wash spin in this protocol. Check which tube you are in before starting the centrifuge.

  1. Discard the supernatant by pouring.
  2. Resuspend the pellet and bring the volume to 40 mL with ice-cold MACS Buffer.
  3. Recap and resuspend by inverting the tube back and forth, or with a brief but firm shake.
  4. Centrifuge at 400 g for 7 minutes at 4 °C (50 mL tube).
  5. Discard the supernatant and keep the leukocyte pellet on ice.
Warning

Cell pellets from digested liver are often loose and poorly compacted.
When discarding supernatant by pouring, do so carefully. Avoid flicking, shaking, or abrupt movements.

Note

Optional hepatocyte pre-clearing spin (recommended). A low-speed spin can be inserted immediately after filtration (before step 6) to sediment hepatocytes while leaving leukocytes in suspension:

  1. Centrifuge at 50 g for 2 minutes at 4 °C.
  2. Carefully transfer the supernatant (containing leukocytes) to a fresh 50 mL tube, avoiding the hepatocyte pellet.
  3. Continue from step 5 above.

Why this is worth doing. Hepatocytes are large, fragile cells and rupture readily at the higher speeds used for the subsequent wash and Percoll spins. Lysed hepatocytes release DNA and cytoplasmic contents that drive clumping and trap leukocytes, and they release damage-associated molecular patterns — extracellular ATP, HMGB1, mitochondrial DNA — which ligate purinergic and pattern-recognition receptors and can activate the very leukocytes being isolated, altering activation-marker expression before staining. Removing intact hepatocytes at low speed first avoids generating that debris.

Weigh this against the leukocyte loss in the discarded pellet: a proportion of leukocytes will be carried down with the hepatocytes. The Percoll gradient alone removes hepatocytes effectively, so this step is optional — but it is recommended where activation phenotype is a readout, where clumping has been a problem, or for single-cell applications.

STEP 6 – Percoll enrichment

Note

The Percoll working solution is 37% of isotonic Percoll stock, which is the same solution as “33% Percoll” when quoted by final Percoll content (37 x 0.9 = 33.3). The AG Surace SOP #001 uses the latter convention. See BUF-PERC-37 — do not relabel one as the other without changing the recipe.

  1. Aliquot 25 mL of 37% Percoll into pre-labelled 50 mL conical tubes (one tube per liver).
Warning

Use a 50 mL tube for the Percoll step, not a 15 mL tube.
In a 50 mL tube the hepatocyte layer floats clear of the pellet and the supernatant can be poured off cleanly.
If a 15 mL tube must be used, the hepatocyte layer sits much closer to the pellet — you must aspirate the supernatant under vacuum rather than pour, or hepatocyte contamination of the leukocyte fraction will be unacceptably high.

  1. Using a transfer pipette, resuspend the cell pellet using the small remaining dead volume.
  2. Take up approximately 1 mL of 37% Percoll from the corresponding 50 mL tube and use it to rinse the wash tube, resuspending any remaining cells.
  3. Transfer the resuspended cells into the corresponding tube containing 25 mL 37% Percoll.
  4. Repeat the rinse 1–2 times with additional small volumes of Percoll (as needed) to recover all remaining cells.
  5. Recap and invert gently 2–3 times to ensure an even cell suspension.
  6. Centrifuge at 611 g for 15 minutes at room temperature (20–22 °C) using:
    • Acceleration: high (e.g. acc 9)
    • Brake: minimal (e.g. br 1)
Note

611 g corresponds to 1700 rpm on the lab centrifuge (rotor rmax ~18.9 cm). If using a different rotor, set the instrument by relative centrifugal force (611 g), not by rpm. Equivalent speeds:

Rotor rmax rpm for 611 g
16.0 cm 1848
17.0 cm 1793
18.0 cm 1742
18.9 cm 1700
20.0 cm 1653

Calculated as RCF = 1.118e-5 x r(cm) x rpm^2.

Warning

Avoid high brake settings during Percoll centrifugation. Abrupt braking can disrupt the gradient and reduce lymphocyte enrichment.

Important

Do not leave liver samples in Percoll longer than necessary. Prolonged exposure can reduce viability and negatively impact downstream applications. Plan spins and next steps in advance.

Note

After the Percoll spin, a hepatocyte layer floats on top of the solution, while a leukocyte + erythrocyte pellet forms at the apex of the tube.

  1. Prepare a few layers of paper towel next to the waste container.
  2. Pour off the hepatocyte layer and supernatant in one smooth motion.
  3. Keep the tube inverted briefly and dab the opening on the paper towel to wick residual Percoll (avoid touching or dislodging the pellet).

Proceed directly to STEP 7 – RBC lysis. There is no intermediate wash.

STEP 7 – RBC lysis

  1. Using a 5 mL or 10 mL Stripette, add 1 mL ACK lysis buffer to each pellet.
  2. Using a transfer pipette, resuspend the leukocyte + erythrocyte pellet by aspirating and dispensing until homogeneous.
  3. Incubate for 2 minutes at room temperature.
  4. During the incubation, transfer each suspension into the corresponding labelled 15 mL tube.
  5. At the end of the incubation, quench by topping up to 10 mL with MACS Buffer.
  6. Centrifuge at 400 g for 5 minutes at 4 °C (15 mL tube; return brake to a normal setting, e.g. br 6).
  7. Discard the supernatant by pouring, dab the inverted tube on paper towel, and keep the pellet on ice.
Warning

Liver leukocytes are particularly sensitive to over-lysis. Do not exceed the stated incubation time, especially after enzymatic digestion or Percoll separation.

Note

HybriMax may be substituted for ACK (2–3 minutes at room temperature) and is gentler, but ACK at 2 minutes is the standard for this protocol.

STEP 8 – Final resuspension and counting

  1. Loosen the pellet in the remaining dead volume by vortexing briefly.
  2. Resuspend the cell pellet in 5 mL ice-cold MACS Buffer.
  3. Count cells and assess viability (e.g. trypan blue) as required.
  4. Proceed with downstream applications (e.g. flow cytometry, sorting, or culture of NLCs).
Tip

For flow cytometry staining, seed up to 5 x 10^6 cells per well of a 96-well U-bottom plate. This is the maximum recommended for a 50 uL staining volume — scale the staining volume up for more cells. Maximum well volume is 200 uL.

Tip

For single-cell sequencing applications, minimise the time from tissue harvest to final resuspension, keep samples on ice whenever possible after digestion, and consider additional debris/dead cell removal steps if yields permit.

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.

Liver Digestion Buffer

Component Final concentration / amount
Collagenase D 1 mg/mL (50 uL of 100 mg/mL stock per 5 mL)
Collagenase IV 0.5 mg/mL (25 uL of 100 mg/mL stock per 5 mL) - alternative to Collagenase D
DNase I 50 ug/mL (25 uL of 10 mg/mL stock per 5 mL)
  • Final volume: 5 mL per liver
  • Preparation note: Mix gently by inversion; do not vortex. Pre-warm to 37C immediately before use.
  • Storage: Keep on ice until pre-warming; do not freeze after DNase I is added; discard unused buffer at end of day.
  • Caveat: Collagenase D and IV are not interchangeable at the same concentration - use one or the other, not both. Never use an EDTA-containing base (e.g. MACS Buffer) - EDTA inhibits collagenase digestion.

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

Percoll Solution (37% of isotonic stock = 33% final)

Component Final concentration / amount
Percoll (isotonic) 9.25 mL (37% of isotonic stock = 33% final Percoll)
PBS 1x 15.75 mL
  • Final volume: 25 mL per mouse liver
  • Preparation note: Equilibrate isotonic Percoll stock to room temperature before diluting; mix gently by inversion. Prepare fresh on the day of use.
  • Storage: Room temperature, same-day use only; do not freeze working solution
  • Caveat: 37% (of isotonic stock) and 33% (final Percoll) both describe this same solution, just measured against different denominators - check which convention an external protocol uses before changing any number.

Full composition, preparation steps, storage, and safety information: BUF-PERC-37.

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 Notes
PBS 1x or FACS Buffer, ice-cold Various BUF-FACS-BUF; perfusion only — never perfuse with MACS Buffer
MACS Buffer In-house / various BUF-MACS-BUF; collection, washes, and final resuspension; kept cold
Liver digestion buffer In-house BUF-LIV-DIG; DMEM, PBS, or HBSS base; collagenase + DNase I; pre-warmed to 37 °C
Collagenase D or Collagenase IV Roche / various Component of liver digestion buffer
DNase I Various Component of liver digestion buffer; 50 ug/mL final
Percoll Cytiva/GE or similar BUF-PERC-37; 37% of isotonic stock = 33% final Percoll; prepared on day of use
ACK lysis buffer Commercial or in-house BUF-ACK-LYSIS; 1 mL per liver; 2 min at room temperature
Fetal calf serum (FCS), heat-inactivated Various Component of MACS Buffer
EDTA Various Component of MACS Buffer
CO₂ or approved euthanasia agent Facility supply For mouse sacrifice

Disposables

Item Notes
50 mL conical tubes Liver collection, filtration, washing, and Percoll enrichment (2 per liver)
15 mL conical tubes Post-lysis wash (one per liver)
gentleMACS C-tubes (or equivalent) Enzymatic and mechanical dissociation
Sterile Petri dishes (10 cm) Backup mincing / straining if required
70 µm cell strainers Filtration of digested tissue
Syringes (5–10 mL) Liver perfusion
Needles (25–27 G) Inferior vena cava or portal vein perfusion
Transfer pipettes Resuspension and Percoll transfer
Filter paper Blotting liver prior to weighing
Paper towels / bench wipes Wicking supernatants after Percoll
Gloves and sharps container Standard laboratory safety

Equipment

Equipment Notes
CO₂ chamber or euthanasia setup As per animal ethics approval
Dissection instruments Scissors and forceps
gentleMACS dissociator With liver-appropriate digestion program
Refrigerated centrifuge Compatible with 15 mL and 50 mL tubes; adjustable brake
Benchtop incubator or heating sleeve 37 °C for enzymatic digestion
Analytical balance For weighing mice and livers
Biological safety cabinet (Class II) Recommended for tissue and cell handling
Ice bucket To keep samples cold between steps
Timer Critical for digestion, Percoll, and lysis steps

Troubleshooting

Problem Possible cause Suggested solution
Low lymphocyte yield Incomplete perfusion or digestion Ensure liver blanches during perfusion; mince thoroughly; avoid under-digestion
High red blood cell contamination Insufficient or skipped RBC lysis Perform ACK lysis for the full 2 min and quench immediately
Poor Percoll separation or weak pellet Incorrect Percoll concentration or harsh braking Use fresh 37% Percoll; confirm 611 g; set brake to minimum
Heavy hepatocyte carryover Percoll run in a 15 mL tube, or supernatant poured Use a 50 mL tube; if 15 mL is unavoidable, vacuum-aspirate the supernatant; consider the optional 50 g pre-clearing spin
Poor digestion despite correct enzyme Perfused with EDTA-containing MACS Buffer Perfuse with PBS or FACS Buffer only
Loose pellets and cell loss Aggressive pouring of supernatant Pour slowly in one smooth motion; wick residual liquid gently
Cells still in supernatant after a wash 50 mL tube spun for the 15 mL duration Spin 50 mL tubes at 400 g for 7 min, 15 mL tubes for 5 min
Low viability Over-digestion or prolonged handling at RT Limit digestion time; keep washes cold; process promptly
Excess debris or clumping Inadequate DNase activity or filtration Confirm DNase at 50 ug/mL; filter through 70 µm mesh with the plunger

Safety (brief)

  • All animal procedures must comply with approved animal licences and institutional regulations.
  • Wear appropriate PPE (lab coat, gloves, eye protection) when handling animals, tissues, and reagents.
  • Handle sharps (needles, scalpels) with care and dispose of immediately in approved sharps containers.
  • Perform tissue and cell handling in a biological safety cabinet where appropriate.
  • Percoll and RBC lysis buffers are chemical hazards; avoid skin and eye contact and dispose of waste according to local safety guidelines.

Version history

Version Date Author Change summary
v1.0 2025-11-20 Dillon Corvino Initial Quarto version based on SOP_001 (liver) from IEO
v1.1 2025-11-26 Dillon Corvino Manual check and update protocol
v1.2 2025-12-29 Dillon Corvino Unified with the established lab protocol
v2.0 2026-08-05 Dillon Corvino Merged with AG Surace SOP #001 (S. Ng). Perfusion switched to ice-cold PBS (EDTA-free) with rationale callout. Mandatory 70 um syringe-plunger filtration directly into a 50 mL tube after gentleMACS. 50 g hepatocyte spin demoted to optional. Two 40 mL washes at 400 g. All 50 mL tube wash spins 5 -> 7 min (15 mL remain 5 min). Percoll: density unchanged (37% of isotonic stock = 33% final Percoll, now labelled with both conventions); 10 mL in 15 mL tube -> 25 mL in 50 mL tube, 800 g/20 min -> 611 g/15 min, acc 7 -> acc 9; post-Percoll wash removed. ACK lysis standardised to 1 mL for 2 min. Final resuspension 5 mL. Digestion buffer consolidated to single BUF-LIV-DIG.
v2.1 2026-09-18 Dillon Corvino Removed the inline ## Buffer recipes section (full recipes were duplicated from the linked buffer documents); replaced # Buffers used with an auto-generated # Buffers Required section (one heading + one small table per buffer, sourced from each buffer’s own summary: data). BUF-FACS-MACS split into BUF-FACS-BUF and BUF-MACS-BUF; reagent references updated accordingly.