May 20, 2026
Polyamine modulation through IP injections
- 1KU Leuven
Protocol Citation: Ana Catarina Carapinha Cascalho, Hanne Dhondt, Nina Schoonvliet, Aisha Sati 2026. Polyamine modulation through IP injections . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8bw6l5r/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: 96901
Keywords: polyamine modulation through ip injection, polyamine modulation, dfmo ip, modulating ip therapy, ip injection, ip therapy, positivie polyamine, spermidine, dfmo, injection, spermine, protocol
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-024297
Abstract
This protocol details the negative (DFMO IP and DenSPM) and positivie polyamine (spermine and spermidine) modulating IP therapy and injections.
Attachments
Materials
Material
- Analytical scale
- Falcon tubes
- 0.22 µm filter (Carl Roth; SE2M035I07)
- Syringe
- 1.5 ml tubes
- Insulin syringes (BD; 324826)
- Scale (precision 0.1 g) to weight the pups
- Tattoo ink (Ketchum; 329AA) + 30 G needle (BD; 304000)
Reagents
- DFMO (CAS: 96020-91-6)
- 0.9 % saline (VWR; 601780ZA)
- N1,N11-Diethylnorspermine tetrahydrochloride (DenSPM; Torcis, CAS 156886-85-0)
- 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
6m
DFMO IP preparation
Calculate the amount of substance to weight depending on the solvent volume you add.
- Formula:
- Stock Concentration:
| A | B | C | D | |
| Age of pups | conc | conc | conc | |
| P0-P5 (1-2.5g) | 0.013g/ml | 13 mg/mL | 13 µg/µL | |
| P6-P14 (2.5-4.5g) | 0.025g/ml | 25 mg/mL | 25 µg/µL | |
| P15-P21 (4.5-8g) | 0.025g/ml | 25 mg/mL | 25 µg/µL |
DFMO (CAS: 96020-91-6) is stored at -20 °C . Weigh the calculated amount. Seal the original compound container with parafilm.
- Write down the opening date if the compound is new.
- Register on your lab book notes the Ref# and Lot# of the prepared stock.
Add with a pipet the correct volume of 0.9 % saline (VWR; 601780ZA).
To 10mL of saline add 0.13 grams of DFMO → 13 mg/mL
To 10mL of saline add 0.25 grams of DFMO → 25 mg/mL
Mix it gently by inverting.
When DFMO is dissolved completely, filter the solution with a 0.22 µm filter (Carl Roth; SE2M035I07) connected to a syringe.
Aliquotes of a pre-defined volume are made and labeled with:
- name and concentration of the solution (DFMO 13 or 25 mg/ml; 1X stock)
- date of preparation
- name of the lab technician
The left over volume is aliquoted in a separate tube with a label mentioning the specific volume.
Store the aliquotes in a box at -20 °C . Keep the solubilized stocks up to 6 months at -20 °C and up to 12 months at -80 °C .
Transfer all the stock preparation information to the lab book.
DFMO IP injections
Day 0: Mice are born
Day 2:
- Check if the litter is still alive and prepare the injection solution for drug delivery.
- Paw tattoo for identification – Update excel sheets for health/behaviour monitorization.
Daily from P2-P21: Inject all pups – Pups should be injected at roughly the same time every day.
IP injection (Vehicle Saline 0.9%):
- Stock Concentration:
| A | B | C | D | |
| Age of pups | conc | conc | conc | |
| P0-P5 (1-2.5g) | 0.013g/ml | 13 mg/mL | 13 µg/µL | |
| P6-P14 (2.5-4.5g) | 0.025g/ml | 25 mg/mL | 25 µg/µL | |
| P15-P21 (4.5-8g) | 0.025g/ml | 25 mg/mL | 25 µg/µL |
- Working Concentration: P0-5 (350 mg/kg); P6-8 (400 mg/kg); P9-P21 (500 mg/kg).
| A | B | C | D | |
| Daily Injections: MAX 40ul/g | example | |||
| P0-P5 | 350 | µg/g | Weight 2 g: 53.84 µL | |
| P6-P14 | 400 | µg/g | Weight 4 g: 64 µL | |
| P15-P21 | 500 | µg/g | Weight 6 g: 120 µL |
- Day 4/7/9/11/14/17/21/every week:
- Register the weight of all mice.
- After weaning (day 21), mice receive DFMO treatment via the drinking water.
DenSPM IP preparation
Calculate the amount of substance to weigh depending on the solvent volume you add (Final concentration = 1.5 mg/mL ).
Formula:
The pure N1,N11-Diethylnorspermine tetrahydrochloride (DenSPM; Torcis, CAS 156886-85-0) powder stocks are stored at Room temperature . Weigh the necessary amount of DenSPM in the appropriate recipient. Seal the compound container with parafilm.
- Write down the opening date if the compound is new.
- Register on your lab book notes the Ref# and Lot# of the prepared stock.
Add with a pipet the correct volume of 0.9 % saline.
Mix it gently by inverting.
When DenSPM is dissolved completely, filter the solution with a 0.22 µm filter connected to a syringe.
Aliquotes of a pre-defined volume are made and labeled with:
- name and concentration of the solution (DenSPM 1.5 mg/ml)
- date of preparation
- name of the lab technician
Transfer aliquots to a box at -20 °C . Keep the solubilized stocks up to 1 month at -20 °C and up to 6 months at -80 °C .
Transfer all the stock preparation information to the lab book.
DenSPM IP injections
Day 0: Mice are born.
Day 2:
- Check if the litter is still alive prepare injection solution for drug delivery.
- Paw tattoo for identification – Update excel sheets for health/behaviour monitorization.
Daily from P2-P42: Inject all pups – Pups should be injected at roughly the same time every day.
- Stock = 1,5 µg/L
- Injection = 60 µg/g or 40 µl/g
- Example: weight: 2 g inject 80 µL
Day 4/7/9/11/14/17/21/every week:
- Register weight of all mice.
POSITIVE POLYAMINE MODULATION
10s
Spermine and spermidine IP preparation
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)
Formula:
Example
Spermine and spermidine are stored at 4 °C . Weigh the calculated amount of spermine or spermidine in the appropriate recipient to reach a concentration of 25 mg/mL . Seal the compound container with parafilm.
- Write down the opening date if the compound is new.
- Register on your labbook notes the Ref# and Lot# of the prepared stock
Add with a pipet the correct volume of 0.9 % saline.
Mix it gently by inverting.
Dissolve the spermine or spermidine completely. Connect a syringe to a 0.22 µm filter to filter the solution.
Make aliquotes of a pre-defined volume and label with:
- name and concentration of the solution (spermine or spermidine 25 mg/ml; 10X stock)
- date of preparation
- name of the lab technician
Aliquoted the left over volume in a separate tube with a label mentioning 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.
Store the aliquotes in a box at -20 °C . Keep the solubilized stocks up to 1 month at -20 °C and up to 6 months at -80 °C .
Transfer all the stock preparation information to the lab book.
Prepare the 2.5 mg/mL working solution by diluting the stock solution 1:10 (example: 1 ml stock solution + 9 ml 0.9 % saline).
IP injection Spermine and Spermidine
Prepare compound fresh every changing day from -20 stock tubes.
Day 0: Mice are born.
Day 2:
- Check if the litter is still alive prepare injection solution for drug delivery.
- Paw tattoo for identification and take tail tissue for genotyping.
Daily from P2-P14: Inject all pups 40 µl/g for SPD and 20 µl/g for SPM for the lowered SPM injections. Pups should be injected at roughly the same time every day.
Day 14:
- Blood collection via the facial vein.
- Collect brain tissue (2 x 1 half hemisphere).