May 20, 2026

Polyamine modulation in drinking water

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzjmb2lx1/v1
  • 1KU Leuven
Hanne HD Dhondt
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzjmb2lx1/v1
Protocol Citation: Ana Catarina Carapinha Cascalho, Hanne Dhondt, Nina Schoonvliet, Aisha Sati 2026. Polyamine modulation in drinking water. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjmb2lx1/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: March 14, 2024
Last Modified: May 26, 2026
Protocol  Integer ID: 96902
Keywords: polyamine modulation in drinking water, polyamine modulation, spermidine, spermine, drinking water preparation, drinking water, dfmo
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-024297
Abstract
This protocol details the DFMO, spermine and spermidine drinking water preparation and administration.
Attachments
Icon for Preparation and administration of polyamine modulating therapies.docx
Preparation and admi...
33KB
Materials
Materials
  • 0.20 µm filter unit (Fisher Scientific; 15933307)
  • Analytical scale
  • Scale (precision 0.1 g) to weigh the drinking bottles
  • 0.22 µm filter (Carl Roth; SE2M035I07)
  • Syringe
Reagents
  • DFMO (CAS:96020-91-6)
  • Spermine tetrahydrochloride; (Thermo Scientific; J63060; CAS: 306-67-2; MW: 348.18 g/mol) (Spermine)
  • Spermidine trihydrochloride (Thermo Scientific; 215100250; CAS: 334-50-9; MW: 254.63 g/mol) (Spermidine)
  • Nitrogen gas

NEGATIVE POLYAMINE MODULATION
2w
DFMO drinking water preparation 
Take DFMO (CAS:96020-91-6) from -20 °C stock.

Drinking water working concentration is 2 Mass / % volume in drinking water (20 g /1 L ).

Example: Dissolve 20 g DFMO in 1 L water.

Weigh the right amount of DFMO.
Dissolve the weighed DFMO in the correct volume of water.
Mix it by gently inverting and wait for the dissolution of the substance in water.
Filter the drinking water through a 0.20 µm filter unit (Fisher Scientific; 15933307).
Store the drinking water at 4 °C . Renew the drinking bottles every 2 weeks.
DFMO drinking water administration
Day 21:

Wean and separate male and female pups (no single mice to avoid stress/anxiety). Pups are then moved to drinking water delivery of compound. Drinking bottles are weighted to monitor the amount of consumed water.


Note
Do not forget to make a clear card and note on bottle so animal caretakers do not change the water in the facility

POSITIVE POLYAMINE MODULATION
2w 0d 0h 0m 10s
Spermine tetrahydrochloride; (Thermo Scientific; J63060; CAS: 306-67-2; MW: 348.18 g/mol) (Spermine)
Spermidine trihydrochloride (Thermo Scientific; 215100250; CAS: 334-50-9; MW: 254.63 g/mol) (Spermidine)
Calculate the amount of substance, depending on the solvent volume, you have to add to reach a concentration of 0.75 Molarity (M) .


Formula:



Example:

I need 25 mL of  0.75 M Spermidine Trihydrochloride stock. (Molecular Weight = 254.63 g/mol).



The pure powder stocks are stored at 4 °C. Weigh the necessary amount with a precision scale, in the appropriate recipient. Seal the container with parafilm.

  • Write down the opening date if the compound was new.
  • Register on the lab book the open date, Ref# and Lot# of every stock solution prepared.
Add with a pro-pipettor a volume of solvent.
Mix it (by gently inverting) and wait for the dissolution of the substance in water.
Filter the solution into a fresh labelled falcon using a 0.22 µm filter (Carl Roth; SE2M035I07) connected to a syringe.
Prepare and label aliquoting tubes:

  • name and concentration of the solution (Spermidine or spermine 25 mg/ml; 10X stock)
  • date of preparation
  • name of the lab technician
Aliquot the solution using the pre-defined volumes:

  1. If preweaning treatment: 400 µL /aliquot
  2. If postweaning treatments: 4 mL /aliquot

Note
If there is leftover volume aliquot it into a separate tube and clearly label the specific volume.


Under the hood, you are going to replace the air into a nitrogen atmosphere to reduce amine degradation.
Carefully open the lid of the tube, place the pipette tip into the tube on the side without touching the liquid. Wait for 00:00:10 . Carefully and quickly close the cap. Repeat for all aliquots.
10s
Discard the pipette tip.
Transfer aliquots to a box at -20 °C . Keep the spermine and spermidine stocks up to 3 months.

Check if you filled in all the information in the template stock preparation, which must be present in the lab book.
Prepare the drinking water for the mice by diluting the 0.75 Mass Percent stock 1:250 (4 mL stock in 1 L tap water). Filter the drinking water through a 0.20 µm filter unit. Store the drinking water at 4 °C Drinking bottles should be renewed every 2 weeks.