JNAR Protocol

Macrophage Isolation from Lung Tissue

Núria Crua· Postdoc· University of Lausanne (UNIL)

Published Jul 27, 2026

~1h 45m total· 2 contributions

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JNAR-ID: JNAR-91788110

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PublishedLast updated Jul 27, 2026

Objective

To isolate lung macrophages from mouse lung tissue using enzymatic digestion, Percoll gradient separation, magnetic bead-based enrichment with anti-F4/80 microbeads.

Classification

Sample typeTissue

TechniqueFlow Cytometry / FACS

Experimental contextEx vivo

Biological modelMouse

Reproducibility status

Not reproducible

1 step blocks the protocol — it can't be completed end-to-end until it is fixed.

38 healthy1 blocking

Step 39Residual F4/80⁺ CD64⁺ Population in the Negative ControlGo to step

⏱ Total 1 h 45 min2 sections39 steps

Lung Tissue Digestion and Single Cell Suspension Preparation

1 h 10 min

Prepare digestion mix:

Reagent	Quantity
Collagenase	1 mg/mL
DNAse I	0.1 mg/mL
RPMI	20 mL

Add 2.5 mL digestion mix into a Gentle MACS Tube and keep on ice.

Place lung tissue pieces into the Gentle MACS Tube containing digestion mix.

Run the appropriate tissue dissociation program on the gentleMACS™ Dissociator.30 min

Place a MACS Smart Strainer on top of a 15 mL tube.

Transfer the digested sample onto the cell strainer using a 5 mL pipette.

Gently mash the tissue remaining on the strainer using a 5 mL syringe plunger to maximize cell yield.

Critical stepExcessive force may damage cells; insufficient mashing reduces yield.

Wash the strainer with ice-cold FACS buffer to deactivate collagenase.

Critical stepFailure to deactivate collagenase may lead to continued digestion and cell death.

Centrifuge at 400g for 5 minutes at 4°C. Discard the supernatant.5 min

Resuspend the pellet in 2 mL RBC lysis buffer. Incubate for exactly 5 minutes at room temperature.5 min

Critical stepOver-incubation may lyse leukocytes; under-incubation leaves RBCs.

Add 10 mL RPMI to stop lysis reaction.

Centrifuge at 400g for 5 minutes at 4°C. Discard supernatant.5 min

Repeat RBC lysis step if red cells remain in pellet; otherwise proceed.

Resuspend pellet in 5 mL 40% Percoll.

Prepare tubes with 5 mL 80% Percoll.

Layer the 40% Percoll-cell suspension gently on top of the 80% Percoll. Do not let it sit.

Critical stepDisturbing the interface or letting it sit reduces separation efficiency.

Centrifuge at room temperature, 1600g for 15 minutes with brake=1 and acceleration=9.15 min

Critical stepIncorrect settings may cause poor layering or cell loss.

Collect the lung cells from the cloudy interface between the 40% and 80% Percoll layers using a 1 mL pipette into new 15 mL tubes.

Add up to 10 mL cold PBS and centrifuge at 500g for 5 minutes at 4°C.5 min

Discard supernatant. Resuspend pellet in 5 mL cold PBS and centrifuge again at 500g for 5 minutes at 4°C.5 min

Count cells

Anti-F4/80 Microbead-Based Enrichment

35 min

Centrifuge lung cell tubes at 300g for 10 minutes at 4°C.10 min

Prepare two 15 mL tubes for each mouse: one for positive selection (+) and one for negative control (-).

Place the negative control tube under the MACS column.

Remove supernatant carefully. Measure remaining media volume after decanting, mix pellet with residual supernatant, and calculate correct buffer volume for resuspension.

Resuspend pellet in the correct volume of buffer.

Vortex microbead vial for 5-10 seconds before use.

Add the correct volume of anti-F4/80 microbeads for each mouse sample.

Mix well and incubate for 15 minutes at 4°C (in fridge).15 min

Wash cells by adding 1-2 mL buffer per 10^7 cells.

Centrifuge at 300g for 10 minutes at 4°C.10 min

During the 10-minute incubation, prepare magnets and columns. Five minutes before incubation ends, wash the column with 3 mL buffer (keep the flow-through, which contains unlabeled cells).

Remove supernatant completely and resuspend pellet in 500 µL buffer (max 10^8 cells; add more buffer if cell count exceeds this).

Critical stepOverloading the column reduces purity and yield.

Apply cell suspension onto the column (keep the flow-through).

Critical stepApply slowly to avoid clogging and ensure bead-bound cells are retained.

Wash column with 3 x 3 mL buffer (keep the flow-through).

Critical stepWashing removes unbound cells; insufficient wash reduces purity.

Remove the tube from the magnet and place the column on a new 15 mL tube (positive tube).

Add 5 mL MACS buffer to the column.

Place the plunger into the column and push strongly and fast to elute the magnetically labeled cells.

Critical stepFast, strong elution ensures maximum recovery of labeled cells.

Count cells and the isolated cells are now ready for downstream applications.

BlockerResidual F4/80⁺ CD64⁺ Population in the Negative Control

The negative control still shows a clearly identifiable F4/80⁺ CD64⁺ population.

Reported by Núria Crua · Jul 27