TITLE: Push-pull microdialysis sampling protocol
AUTHORS: Christiana.bjorkli  

[DESCRIPTION]
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. 

[STEPS]
SECTION: Setting up the system
2. Set a refrigerated fraction collector (CMA 470) to6 °C for the storage of collected CSF in polypropylene plastic vials.

SECTION: Setting up the system
3. 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).

SECTION: Setting up the system
4. 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.
SECTION: Setting up the system
5. Connect FEP tubing (CMA Microdialysis AB, Kista, Sweden) to each microsyringe. Then connect FEP tubing to the inlet part of the microdialysis probes.
SECTION: Setting up the system
6. Prepare transparent cages with 1.5 cm of bedding, filled water bottles, and treats.
SECTION: Setting up the system
7. 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).
SECTION: Habituation of animals
1. Habituate mice to human handling and restraining for one week prior to microdialysis sampling.
SECTION: In vivo Microdialysis
10. 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. 

SECTION: After the experiment
12. At the conclusion of microdialyte sampling,  centrifuge 100 rpm vials of 60 µL CSF  and keep at -80 °C until further analyses.

SECTION: After the experiment
13. Discard all tubing after the experiment. 
SECTION: Setting up the system
8. Calculate the ‘dead volume’ of the FEP outlet tubing (1.2mL/100mm). Use 100 cm of FEP outlet tubing, and thereby the first 12 µL of CSF sampled from each animal should be discarded. 
SECTION: In vivo Microdialysis
11. Set the flow rate on the syringe and peristaltic pump to 0.2 µL/min for 72 min. 

SECTION: Setting up the system
9. Run the syringe and peristaltic pump at 60 µL/min to fill the entire system with artificial CSF + BSA solution.