Apr 29, 2026
Immune cell isolation from mouse brain and spleen for flow cytometry
- Amandine EVEN1,2,3,4,5,
- Sriparna Mukherjee1,2,3,5,
- Louis-Éric Trudeau1,6,2,3,4,5
- 1Department of pharmacology and physiology, Faculty of Medicine, Université de Montréal;
- 2Neural Signaling and Circuitry research group (SNC);
- 3Center for Interdisciplinary Research on the Brain and Learning (CIRCA);
- 4Institut Courtois d’innovation biomédicale;
- 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 6Department of neuroscience and physiology, Faculty of Medicine, Université de Montréal
Protocol Citation: Amandine EVEN, Sriparna Mukherjee, Louis-Éric Trudeau 2026. Immune cell isolation from mouse brain and spleen for flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoe68bl4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 20, 2026
Last Modified: April 29, 2026
Protocol Integer ID: 315388
Keywords: immune cell isolation from mouse brain, spleen for flow cytometry, subsequent flow cytometric analysis of immune cell, cell suspensions from the spleen, flow cytometry, vivo cd45 labeling, enzymatic dissociation of mouse brain tissue, immune cell isolation, mouse brain tissue, spleen tissue, immune cell, cytometric analysis, steps for red blood cell lysi, spleen lysate, antibody, cell suspension, spleen, myelin removal, red blood cell lysi, mouse brain, enzymatic dissociation
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000525
Abstract
This protocol outlines the procedures for terminal in vivo CD45 labeling, enzymatic dissociation of mouse brain tissue using collagenase D and DNase I, myelin removal via a Percoll gradient, and mechanical preparation of single-cell suspensions from the spleen. It also includes steps for red blood cell lysis in spleen lysate, antibody staining, fixation, and subsequent flow cytometric analysis of immune cells isolated from both brain and spleen tissues.
Guidelines
**Suggested gating workflow**
I. Begin with FSC-SSC to exclude debris, then gate singlets.
II. Exclude dead cells using viability dye.
III. Use the intravenously delivered CD45-BV711 signal to help distinguish vascular/circulating cells from brain-associated.
IV. Define myeloid and lymphoid compartments using CD45, CD11b, CD11c, Ly6C, Ly6G, F4/80, CD3e, CD4, CD8a, CD19, and NK1.1 as appropriate.
**Quality control**
I. Process all comparison groups in parallel.
II. Avoid prolonged delays between tissue dissociation and staining.
III. Inspect brain suspensions after Percoll cleanup as excessive residual myelin can degrade cytometer performance and data quality.
IV. Maintain consistent cell numbers and antibody incubation times across samples.
Materials
**Buffers and solutions**
- Brain digestion solution**: 2 mg/ml collagenase D + 28 U/ml DNase I
- Dissociation / wash buffer**: PBS with 2 mM EDTA and 2% FBS
- Percoll solution**: 37% (v/v) Percoll (in HBSS 1X)
- RBC lysis buffer**: 0.1 M EDTA, 155 mM NH4Cl, 10 mM KHCO3
- 4% PFA**: PFA dissolved in 1X PBS and pH adjusted to 7.4
**Materials**
- 100 µm Falcon cell strainers
- Glass pipette for intermittent trituration
- Hemocytometer
**Equipments**
- Centrifuge with room-temperature and 4°C settings
- BD LSRFortessa X-20 flow cytometer
- FlowJo software
**List of antibodies**
- CD45-BV711**: Rat, 3ug/mouse, Biolegend, Cat# 103147, RRID:AB 2564383
- CD45-FITC**: Rat, 1:100, Biolegend, Cat# 103107, RRID:AB 312972
- CD11b-BV605**: Rat, 1:200, Biolegend, Cat# 101237, RRID:AB 11126744
- CD11C-PECY7**: Hamster, 1:200, BD Biosciences, Cat# 561022, RRID:AB 2033997
- LY6C-AF700**: Rat, 1:100, BD Biosciences, Cat# 561237, RRID:AB 10612017
- LY6G-BV510**: Rat, 1:100, Biolegend, 1087356
- F4/80-A647**: Rat, 1:200, Biolegend, Cat# 123121, RRID:AB 893492
- CD86-BV650**: Rat, 1:200, Biolegend, Cat# 105035, RRID:AB 11126147
- CD3e-PerCp Cy5.5**: Armenian hamster, 1:200, Biolegend, Cat# 100328, RRID:AB 893318
- CD8a-A647**: Rat, 1:100, Biolegend, Cat# 100724, RRID:AB 389326
- CD4-APC Cy7**: Rat, 1:100, Biolegend, Cat# 100525, RRID:AB 312726
- CD19-BV786**: Rat, 1:100, BD Biosciences, Cat# 563333, RRID:AB 2738141
- NK1.1-BV650**: Mouse, 1:50, Biolegend, Cat# 108735, RRID:AB 11147949
Safety warnings
Avoid harsh handling in appropriate buffer and/or temperature conditions.
Before start
- Prepare the glass pipette for trituration:
Melt the tip of the glass pipette to reduce the size of the hole using a Bunsen burner, rotating the pipette to avoid blocking the hole. Three different hole sizes will be created to allow the tissues to pass through progressively smaller holes.
- Prepare all digestion and staining reagents fresh.
- Pre-cool PBS- EDTA-FBS dissociation buffer, and collection tubes for spleen processing.
- Bring collagenase digestion reagents to working temperature immediately before brain dissociation.
- Plan acquisition order so that all groups are processed comparably across the experiment.
Tissue collection
24m
Anesthetize the mouse with an intraperitoneal injection of pentobarbital at100 mg/kg .
15m
Inject intravenously 3 µg BV711-conjugated anti-CD45 diluted in 50 µL of saline.
1m
Allow 00:03:00 for intravascular labeling prior to terminal collection and cervical dislocation.
3m
Tissue harvesting:
5m
Rapidly remove the brain and mince tissue with a scalpel.
Collect spleen from the same mouse and keep it in ice-cold PBS 1X.
Brain tissue digestion and cell suspension preparation
1h 12m 30s
Digest minced brain tissue:
45m
Add the pieces of tissue in a 15mL tube containing 4mL of collagenase D (2 mg/mL ) and DNase I (28 U/mL ) solution.
Put the tube for agitation in a water bath at 37 °C for a total time of 00:45:00 .
Every 00:15:00 , triturate with a glass pipette, keep cells in a separate tube and add fresh dissociation solution.
Repeat 3 times and reduce the size of the glass pipette opening with each new repetition.
Stop enzyme activity by adding cold PBS containing 2 millimolar (mM) EDTA and 2 % (v/v) FBS.
30s
Pass the dissociated tissue through a 100 µm strainer to remove undigested fragments
1m
Centrifuge the homogenate at Room temperature for 200 x g, Room temperature, 00:02:00 and then for 300 x g, Room temperature, 00:03:00 discarding the supernatant carefully.
5m
Remove myelin:
Resuspend the pellet in 10 mL of 37 % (v/v) Percoll.
This step have to be done with solution kept at Room temperature .
1m
Centrifuge for 500 x g, Room temperature, 00:10:00 with no brake.
10m
Aspirate the myelin layer and wash the cell pellet with PBS-EDTA-FBS buffer.
5m
Count recovered cells using a hemocytometer and proceed to antibody staining.
5m
Spleen tissue dissociation and and cell suspension preparation
34m
Mash the tissue through a 100 µm strainer using the thumb-rest of a syringe plunger.
2m
Rinse the filter several times with PBS-EDTA-FBS dissociation buffer to maximize recovery.
2m
Centrifuge homogenates at 1500 rpm, 4°C, 00:10:00 .
10m
Resuspend splenocytes in 3 mL RBC lysis buffer and incubate for 00:03:00 at Room temperature .
Wash with PBS-EDTA-FBS buffer and centrifuge again for 00:10:00 at 4 °C .
10m
Resuspend in 1 mL dissociation buffer, count cells, and reserve 2 million cells per spleen for antibody staining.
10m
Antibody staining and acquisition
42m
Plate cells:
Perform staining at 4 °C in canonical round-bottom 96-well plates protected from light.
Distribute brain and spleen cells in different plates according to the experiment layout.
Centrifuge the plate at 2500 rpm, 4°C, 00:00:40 and discard the supernatant.
40s
Viability stain:
Resuspend the pellet in fixable viability dye eFluor450 (diluted at 1:1000 in 1X PBS).
Incubate for 00:15:00 .
15m
Wash, centrifuge the plate at 2500 rpm, 4°C, 00:00:40 and discard the supernatant.
40s
Block nonspecific binding using Fc block solution:
Resuspend cells in 50 µL of anti-CD16/32 Fc-blocking solution at 0.5 µg per million cells for 00:15:00 .
15m
Surface staining:
Add 50 µL of the antibody cocktail and incubate for 00:30:00 .
Wash using PBS-EDTA-FBS buffer.
Centrifuge 2500 rpm, 4°C, 00:00:40 , discard supernatant.
40s
Fix cells in 4% PFA (100µL in each well) for 00:10:00 .
10m
Set controls: Prepare FMO controls and compensation controls using OneComp eBeads incubated with fluorescent antibodies.
Acquiring data:
Select a flow cytometer with lasers and detectors compatible with the chosen fluorophores.
Here, data were acquired using a BD LSRFortessa X-20.
Acquire samples using appropriate instrument settings (e.g., voltages and compensation).
Analysis
Analyze using FlowJo software
Evaluated time per step