Carmen Mª Navarrón Izquierdo Jose Lopez-Atalaya’s lab

Fluorescent Microglia Cell Sorting

Context:
The next protocol is indicated for a whole cortex of one adult mouse. Maintain the temperature
of tissue/cells at 4ºC during the whole process.
Materials needed:
- Wheaton Tissue Homogeneizer Tenbroeck (with expanded reservoir and pouring lip) 7mL
capacity (0,23 oz), tube length 82mm, made of borosilicate glass (FisherScientific #10198611).
- Quadro MACS Separator (Miltenyi Biotec #130-090-976)
- MultiStand (Miltenyi Biotec #130-042-303)
- LS Columns (Miltenyi Biotec #130-042-401)
- 50mL tubes 4ºC centrifuge (swing rotor)
- Student Dumont #5 Forceps (FST #91150-20)
- Dissecting Chisel 12,5cm (FST #10095)
- 40um cell strainer
- Glass Pasteur Pipettes.
Buffers needed:
- DOUNCE buffer:
15mM HEPES
0,5% glucose
HBSS1x

- MACS buffer:
0,5% BSA
2mM EDTA
PBS 1x

- FACS buffer:
1% BSA
2mM EDTA
25mM HEPES
PBS1x

- Isotonic Percoll Solution (SIP):

Percoll : PBS10x  9:1
Carmen Mª Navarrón Izquierdo Jose Lopez-Atalaya’s lab

Cell suspension preparation:

1. Dissect the brain of an adult mouse. Put it in sterile ice-cold PBS1X. Remove the
meninges with dissecting forceps and extract the whole cortex (̴ 200 mg).
2. Put the dissected cortex in a plate containing 2mL of dounce buffer and cut it in small
fragments with a dissecting chisel.
3. Transfer to a tissue homogeneizer containing 7mL of dounce buffer. Homogeneize the
tissue with 4-5 strokes.
4. Transfer the cell suspension through a 40um cell strainer over a 50mL tube in ice
containing 0,5mL of dounce buffer. Use the plunger of a syringe to help the cells to
pass through the strainer.
Myelin Removal:
This step can be done with: a) Miltenyi Myelin removal beads; b) Percoll Gradient.
a) Miltenyi myelin removal beads:
5. Centrifuge 800G 10min 4ºC ac/dc 9/9.
6. Remove supernatant and resuspend the pellet in 720uL MACS buffer. Add 60uL of
myeling removal beads and mix by pipetting.
7. Transfer the sample to two tubes of 2mL (400uL in each tube) and incubate 10minute
at 4ºC (inside the fridge, not in ice).
8. Bring volume of tubes with samples to 2mL with MACS buffer.
9. Pellet cells. Centrifuge 30s at 10K rpm at 4ºC.
10. Repeat wash (steps 8 and 9).
11. Resuspend cells in 2mL of MACS buffer per tube.
12. Meanwhile, place 2 LS columns per sample in MACS magnet (Quadro MACS +
multistand) and rinse the column with 2mL of MACS buffer to equilibrate it.
13. Apply cells from one tube into one LS column. LS columns will retain the myelin and
cell suspension will flow through it. We recover the flowthrough in a 50mL tube in ice.
14. Centrifuge the tube 800G 10min 4ºC ac/dc 9/9.
15. Resuspend the pellet in 350uL of FACS buffer.

b) Percoll Gradient:
5. Add 2,5mL of isotonic percoll solution (SIP) in orden to obtain a final percoll
concentration of 25%. Total volume = 10mL.
6. To form 2 phases, add 15mL of PBS1x with a glass Pasteur pipette.
7. Centrifuge 800G 15min 4ºC ac/dc 0/0.
8. Remove the myelin from the interphase together with the rest of the supernatant. (The
percoll is in the lower phase together with the cell pellet, it is important not to aspirate the
pellet, so don’t aspirate the last 2mL of percoll)
9. Add 2mL PBS 1x to the remaining percoll. Resuspend the cell pellet and divide this
volume into two Eppendorf tubes (2mL) and centrifuge 10.000rpm 30s 4ºC.
10. Remove supernatant and add 1mL of PBS1x to each Eppendorf tube. Mix both tubes
(with 1mL each) into a 2mL Eppendorf tube. Centrifuge 10.000rpm 30s 4ºC.
11. Resuspend the pellet in 350uL of FACS buffer.

References related with this protocol:

- New tools for studying microglia in the mouse and human CNS. Bennett M.L., … Barres
B.A. PNAS. March 2016. https://doi.org/10.1073/pnas.1525528113
Carmen Mª Navarrón Izquierdo Jose Lopez-Atalaya’s lab

- Neuronal hyperactivity disturbs ATP microgradients, impairs microglial motility, and

reduces phagocytic receptor expression triggering apoptosis/microglial phagocytosis
uncoupling. Abiega O, … Sierra A. PLoS Biol. May 2016.
https://doi.org/10.1371/journal.pbio.1002466
- The microglial reaction signature revealed by RNAseq from individual mice. Hirbec H., …
Rassendren F. Glia. May 2018. DOI: 10.1002/glia.23295