Oct 08, 2021

Public workspacePush-pull microdialysis sampling protocol

DOI
dx.doi.org/10.17504/protocols.io.bszunf6w
  • 1The Norwegian university of Science and Technology
  • Sandvig lab
Christiana.bjorkli
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DOI: dx.doi.org/10.17504/protocols.io.bszunf6w
Protocol Citation: Christiana.bjorkli 2021. Push-pull microdialysis sampling protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bszunf6w
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: March 04, 2021
Last Modified: October 08, 2021
Protocol Integer ID: 47892
Abstract
Our modified push-pull microdialysis method was first validated ex vivo with human CSF samples, and thenin vivoin in an AD mouse model, permiting assessment of dynamic changes of CSF Aβ and tau and allowing for better translational understanding of CSF biomarkers. At the beginning of our experiments, all microdialysis probes were perfused with artificial CSF with 4 % bovine serum albumin (BSA) at a flow rate of 60mL/min in order to fill the entire system with the perfusate. A 7-day equilibration period was always allowed before starting in vivoCSF sampling.
Materials
Chemicals and solutions
1. Artificial cerebrospinal fluid (aCSF)
2. 30% Bovine serum albumin (BSA) solution (Sigma-Aldrich)
3. 70% ethanol
4. Distilled H2O

Equipment and accessories for microdialysis
  1. Microdialysis probes
  2. Microdialysis syringe pump
  3. Microsyringe for syringe pump
  4. Peristaltic pump
  5. Refrigerated fraction collector
  6. Sample collection vials for refrigerated fraction collector with caps and labels
  7. FEP tubing
  8. Peristaltic tubing with stoppers
  9. Tubing adaptors
  10. Cage for freely moving mice
  11. One-time use filter and syringe


Habituation of animals
Habituation of animals
Habituate mice to human handling and restraining for one week prior to microdialysis sampling.
Setting up the system
Setting up the system
Set a refrigerated fraction collector (CMA 470) toTemperature6 °C for the storage of collected CSF in polypropylene plastic vials.

Place fluorinated ethylene propylene (FEP) peristaltic tubing (CMA Microdialysis AB, Kista, Sweden) inside each plastic vial for collection and connected to the cassette of the peristaltic roller pump (Reglo ICC Digital).

Connect peristaltic FEP tubing to the outlet side of the microdialysis probes (β-irrigated 2 mDa microdialysis probe; CMA 7;CMA Microdialysis AB, Kista, Sweden) with a polyethersulfone 2 mm membrane with tubing adapters bathed in 75% ethanol.
Connect FEP tubing (CMA Microdialysis AB, Kista, Sweden) to each microsyringe. Then connect FEP tubing to the inlet part of the microdialysis probes.
Prepare transparent cages with 1.5 cm of bedding, filled water bottles, and treats.
Use artificial CSF (CMA Microdialysis AB, Kista, Sweden) that consists of NaCl (147 mmol/l), KCl (2.7 mmol/l), CaCl2 (1.2 mmol/l) and MgCl2 (0.85 mmol/l). Mix the artificial CSF with 4 % BSA (30 % in saline; Sigma-Aldrich, Saint-Louis, MO, USA) to increase osmotic pressure of the perfusate to allow for better recovery of proteins. Filter the solution after adding BSA. Make this solution fresh prior to each sampling session. Load artificial CSF inside a gastight microsyringe (CMA Microdialysis AB, Kista, Sweden), and place the microsyringe into the syringe pump (CMA 4004).
Calculate the ‘dead volume’ of the FEP outlet tubing (1.2mL/100mm). Use 100 cm of FEP outlet tubing, and thereby the first Amount12 µL of CSF sampled from each animal should be discarded.
Run the syringe and peristaltic pump at Amount60 µL /min to fill the entire system with artificial CSF + BSA solution.

In vivo Microdialysis
In vivo Microdialysis
Restrain mice in order to remove the dummy probe of the guide cannula and replace it with the microdialysis probe. Prior to inserting the microdialysis probes into the guide cannula, condition the probe in 75% ethanol for better recovery of analytes.

Set the flow rate on the syringe and peristaltic pump to Amount0.2 µL /min for Duration01:12:00 .

1h 12m
After the experiment
After the experiment
At the conclusion of microdialyte sampling, centrifuge Centrifigation100 rpm vials of Amount60 µL CSF and keep at Temperature-80 °C until further analyses.

Discard all tubing after the experiment.