Apr 08, 2026

Collection of mouse cerebrospinal fluid from the cisterna magna

DOI
https://dx.doi.org/10.17504/protocols.io.3byl4818zvo5/v1
  • 1KU Leuven
Hanne HD Dhondt
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl4818zvo5/v1
External link: https://app.jove.com/v/56774/an-improved-method-for-collection-cerebrospinal-fluid-from
Protocol Citation: Aisha Sati, Hanne Dhondt 2026. Collection of mouse cerebrospinal fluid from the cisterna magna. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4818zvo5/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: 244065
Keywords: ASAPCRN, cerebrospinal fluid from the cisterna magna, cerebrospinal fluid, cisterna magna, collection of mouse, mouse
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000458
Abstract
This protocol describes the collection of mouse cerebrospinal fluid from the cisterna magna.
Materials
Materials:

  • Stereotax
  • Surgical microscope with light (or separate light source)
  • CSF-collection KIT:
  1. Surgical scissors
  2. Forceps, straight+kinked
  3. Hooks 2x
  4. Small sponges
  5. Tape
  6. 1ml syringe
  • Anaesthetic
  • 1ml Syringe + injection needle
  • Pulled glass-capillaries ( borosilicate glass OD:1.0 mm, ID: 0.75 mm, 10 cm – B100-75-10, sutter instruments)
  • Settings puller:
  1. HEAT 600
  2. PULL 105
  3. VEL 50
  4. Time 100 - 140
  • Tubing ( Microflex 25G - naald met 1 vleugeltje met verlengslang 30 cm - box van 20 pcs, Vygon ref: 240.05)
  • 0,5 ml Low bind Eppendorf tubes
  • Dry ice
  • Centrifuge (ideally 4°C)
  • Pipette + Tips (5µl)
Preparing pulled glass capillaries and micromanipulator set up for CSF collection:
10m
Prepare pulled glass capillaries using a micropipette puller for piercing through the cisterna magna and collecting CSF.
Mount sharpened a capillary on a micromanipulator then connect the capillary through the other end to thin tubing connected to a syringe for CSF sample collection.
Break the tip of the pulled capillary: grab the tip with the straight forceps in a ca. 40° angle approximately in the middle of the pulled tip. Pay attention that the tip is about 10-20µm.
Push out air with the syringe and fix on descending arm of the stereotaxic apparatus.
Mouse anasthezia and head fixation
Anaesthetize the mouse using the folloiwng cocktail: 0,15ml Nimatek + 0,2ml Domitor + 1,5ml sterile saline via intraperitoneal injections. Dosing 0,1ml per 10g of body weight.
Check anaesthesia effect with toe pinch response.
Once the animal is fully anaesthetized, place the animal on a heating pad, and secure the head in the stereotaxic apparatus with ear bars.

Cut away some of the fur on the dorsal neck region, then make a sagittal incision into skin, on top a small incision to the left and to the right to expose the muscle. Clean the field from blood and hair using small amounts of saline solution on cotton buds.
Exposing the cisterna magna and capillary insertion:
10m
Separate the subcutaneous tissue and the muscles (m. biventer cervicis and m. rectus capitis dorsalis major) by blunt dissection with forceps.

Separate the muscles gently using a forceps until the dura becomes visible. Pay attention not to pinch or destroy the dura.

The exposed cisterna magna with the overlaying muscles retracted to either side

Using the pulled capillary prepared in step 1, pierce the dura of the cisterna magna just so that the tip of the capillary is located in the cavity without touching any tissue or blood vessel.


Site of puncture in the middle of the triangle away from major blood vessels. Site can vary slightly from mouse to mouse.
CSF collection
Carefully, apply a bit of negative pressure by pulling the plunger of the syringe. Observe the inserted capillary the whole time through the microscope.
To collect more CSF, allow the cisterna to refill. When the pressure reaches a normal level again, which is often indicated by the vessel pulsating, slowly and carefully apply some more negative pressure.
To remove the capillary from the cisterna, wait until the pressure reaches a normal level again.
Collet CSF from the capillary into a protein low-binding eppendorf.

Spin CSF at 10000 x g, 4°C, 00:10:00 , then pipet the supernatant into a new protein low-binding eppendorf.

Store samples -80 °C .
Remarks
If the triangle is not well visible, or the CSF is not being collecting into the glass capillary, adjust the angle of the head.
Protocol references
This protocol is kindly shared by Sophie Croes of the Prof. Veerle Baekelandt lab.