Before you start
- Bring Percoll solutions to room temperature.
- Prepare digestion medium fresh.
Reagents and materials
Totals are for 4 mice (+10% extra). Per-mouse (brain + spinal cord) amounts are in the middle column.
| Reagent | Per mouse (brain + spinal cord) | Total |
|---|---|---|
| Cold PBS for perfusion | 20 mL | 20 mL |
| Digestion medium: RPMI or HBSS with collagenase D (1 mg/mL) or collagenase IV + DNase I | 5 mL | 5 mL |
| Isotonic Percoll, 30% (in medium) | 7 mL | 7 mL |
| Isotonic Percoll, 70% | 3 mL | 3 mL |
| FACS buffer (washes) | 20 mL | 20 mL |
Buffers and recipes
Isotonic (100%) Percoll per 10 mL
| Percoll | 9 mL |
| 10× PBS or 10× HBSS | 1 mL |
Dilute this 100% stock with medium or PBS to make 30% and 70%.
FACS buffer per 500 mL
| PBS (Ca²⁺/Mg²⁺-free) | 490 mL |
| Heat-inactivated FBS (2%) | 10 mL |
| 0.5 M EDTA, pH 8.0 (1 mM final) | 1 mL |
Filter (0.22 µm) and keep at 4 °C. 0.5% BSA can replace FBS. Sodium azide (0.05–0.1%) is optional for staining only — never for cells you will culture.
Equipment
- Perfusion set-up (peristaltic pump or syringe, 25–27 G needle)
- Scalpel or scissors for mincing
- 37 °C shaking incubator
- 70 µm strainers
- 15 mL tubes
- Centrifuge with brake control
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Perfuse
Under terminal anaesthesia as approved, perfuse transcardially with 20 mL cold PBS until the liver blanches, to remove blood cells from the CNS.
Dissect
Remove the brain and flush the spinal cord out of the column (hydraulic extrusion with PBS) into cold medium.
Mince and digest
Mince finely and digest in 5 mL digestion medium for 30–45 min at 37 °C with gentle agitation.
Make a single-cell suspension
Pass through a 70 µm strainer with a plunger, rinse, and centrifuge at 400 × g for 5 min.
Build the gradient
Resuspend the pellet in 7 mL 30% Percoll and slowly underlay 3 mL 70% Percoll with a pipette.
Centrifuge
Spin at 500 × g for 20–30 min at room temperature, acceleration low and brake off.
Remove myelin and collect cells
Aspirate the myelin layer from the top, then collect the cells at the 30/70 interface.
Wash and count
Dilute at least 3-fold with FACS buffer, centrifuge at 500 × g for 5 min, resuspend and count.
Critical points
- Good perfusion is essential; otherwise blood leukocytes contaminate the CNS fraction.
- Cell numbers from healthy CNS are small; many more cells are recovered at the peak of EAE.
- Enzymatic digestion can cleave some surface epitopes — check your markers.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Myelin contamination | Remove the top layer carefully; use a larger dilution before the final wash. |
| Few cells | Check perfusion, digestion time and that the interface was collected completely. |
Safety
Terminal procedure under your approved animal protocol.
Further reading
- Pino PA, Cardona AE. Isolation of brain and spinal cord mononuclear cells using Percoll gradients. J Vis Exp (2011).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.