Apr 08, 2026
Microglia isolation using magnetic cell sorting from adult mouse brains
- 1KU Leuven
Protocol Citation: Aisha Sati 2026. Microglia isolation using magnetic cell sorting from adult mouse brains. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo1w19g4o/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: February 24, 2026
Last Modified: April 08, 2026
Protocol Integer ID: 244064
Keywords: ASAPCRN, microglia isolation, isolation of microglia, microglia, magnetic beads from adult mouse brain, adult mouse brain, adult mouse brains this protocol detail, magnetic cell, using magnetic cell, using magnetic bead, magnetic bead
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Abstract
This protocol details the isolation of microglia using magnetic beads from adult mouse brains.
Materials
Solutions:
Enzyme digestion mix made fresh on the day:
- HBSS (without calcium/magnesium),
- Fetal Bovine Serum: 5%,
- HEPES (1 mM stock): 10 μM (1ml stock/100ml solution),
- Collagenase A: 2 mg/mL,
- DNase I (lot dependent-need to check with company: 3181U/mg): 28 U/mL (0.0088mg/ml).
MACS buffer:
- 1X PBS: 1 L,
- EDTA (0.5 M): 1 mM (0.2ml stock/100ml),
- Bovine Serum Albumin: 1%. First, dissolve EDTA and BSA in 500 mL of 1X PBS. Then raise volume to 1 L with 1X PBS. filter slowly for 30 min in tissue culture hood. Keep sterile.
Before start
- Aliquot 5 mL of Enzyme Digestion Mix into 15 mL tubes and weigh tubes.
- Prepare aliquots of PBS and Enzyme digestion mix for the dissection. Around 5ml per brain.
Animal dissection and brain collection
Following transcardial perfusion with 0.9% saline, dissect out brain region of interest on an ice-cold petri dish with forceps and place in cold enzyme digestion mix On ice .
Preparing single cell suspension
25m
Place brain tissue samples in enzyme digestion mix in a 37 °C bath for 00:15:00 : (Agitate every 5 min. Agitate tubes, but do not invert to mix). Meanwhile, pre-cool the centrifuge to 4 °C .
15m
Dissociate tissue to a single-cell suspension using three separate flame-polished Pasteur pipets with narrowing opening: Pipet up and down 20 times with each pipet starting with the largest diameter to narrowest.
Note
Incubate at 37°C for 15 min in between each numbered pipet step (Agitate every 5 min within the 15 min incubation of every pipet).
Filter samples into fresh 15 mL tubes. Place 70 μm nylon filters on top of fresh 15 mL tube. Pre-wet each filter.
Note
Pre-wet each filter by adding 1 mL of HBSS (with calcium/magnesium) to the middle of the filter and then drag to side.
Add cell suspension over the pre-wet area on each filter.
Rinse old 15 mL tubes with 2 mL of HBSS (with calcium/magnesium). Pipet up and down to mix. Add over the filter.
Fill the new tubes to 15 mL with HBSS (with calcium/magnesium).
Note
After this step, samples should be kept on ice as much as possible.
Centrifuge: 300 x g, 4°C, 00:10:00 , max acceleration, half brake.
10m
Removing debris/myelin from single-cell suspension
20m
Aspirate the supernatant, then resuspend cell suspension carefully with 3.1 mL of ice-cold 1X PBS and transfer cell suspension to a fresh 15 mL tube.
Add 900 µL of cold Debris Removal Solution.
Mix well by pipetting 10 times slowly up and down.
Slowly layer 4 mL of 1X PBS on top, creating a visible gradient.
Note
Begin with tube tilted at 45° angle and slowly layer 1X PBS on top of Debris REMOVAL Solution layer. As more 1X PBS is added, slowly bring tube back to a vertical position.
Centrifuge for 00:10:00 , max speed (at least 3000 x g ), 4 °C , max acceleration, max brake.
10m
Remove the top two layers. Fill up to 14 mL with ice cold 1X PBS.
Centrifuge: 1000 x g, 4°C, 00:10:00 , max acceleration, max brake.
10m
Magnetic cell separation
45m
Aspirate the supernatant, then re-suspend pellets with 180 µL MACS buffer and 20 µL CD11b microbeads.
Incubate On ice for 00:15:00 .
15m
Add 1 mL of MACS Buffer to samples.
Centrifuge: 300 x g, 4°C, 00:10:00 .
Note
While samples are spinning, set up sorting columns in tissue culture hood.
10m
Place the columns tightly in the magnet (must be fully in place). Then place the “-” tubes that will be used to collect CD11b negative cells under the columns.
Note
Top the columns with fresh nylon filters using autoclaved forceps to maintain sterility.
Prewash each column with 3 mL of MACS Buffer through nylon filter.
Note
Pre-wet each filter by adding 1 mL of HBSS (with calcium/magnesium) to the middle of the filter and then drag to side.
Remove samples from centrifuge and aspirate the supernatant. Resuspend pellets in 500 µL of MACS Buffer.
Add cell suspension to nylon filter, and then rinse the old 15 mL tube with 2 mL of MACS Buffer and add it to the nylon filter.
Add 3 mL of MACS Buffer to column.
Once liquid has finished flowing through the columns, remove the “-” tubes and place On ice .
Remove the columns from the magnet, and place on top of “+” tubes that will be used to collect CD11b positive cells.
Using a serological pipet, add 5 mL of MACS Buffer to the top of the column, and then quickly plunge column as hard as possible using the supplied plunger. Repeat this (for a total of 10 mL volume) for each column. Then place “+” tubes on ice with the “-” tubes.
Centrifuge the “+”and “-” tubes at 300 x g, 4°C, 00:10:00 .
10m
Aspirate the supernatant (make sure to remove all bubbles, especially those from the “+” tubes).
Note
At this point you will only see a robust pellet in the “-” tubes and will not likely see a pellet in the “+” 15 mL falcon tubes. Leave a meniscus of liquid covering the bottom of the tube prior to washing the pellet in 1X PBS, to avoid cell loss.
Resuspend pellet in 400 µL of ice-cold 1X PBS.
Transfer the 400 µL of cell resuspension to fresh 1.5 mL centrifuge tubes, rinse the old 15 mL tube with 800 µL of 1X PBS, and add this rinse to the new 1.5 mL tube as well.
Centrifuge: 300 x g, 4°C, 00:10:00 .
10m
Carefully remove supernatant and resuspend in a lysis buffer (RLT, Qiagen, 74004) and stored
at -80 °C until processing for qPCR.
Protocol references
Adapted from: