TITLE: Sonication of PFFs for use in vitro
AUTHORS: Vijay Singh, Marta Castellana-Cruz, Nunilo Cremades, Laura Volpicelli-Daley

[DESCRIPTION]
Animal models that accurately recapitulate the accumulation of alpha-synuclein (α-syn) inclusions, progressive neurodegeneration of the nigrostriatal system and motor deficits can be useful tools for Parkinson's disease (PD) research. The preformed fibril (PFF) synucleinopathy model in rodents generally displays these PD-relevant features, however, the magnitude and predictability of these events is far from established. We therefore have optimized the synthesis generation of α-syn fibrils to ensure reliable, robust results. These fibrils can be added to neurons in culture, differentiated iPSCs, or injected into mice or rats. The protocol includes steps for fibril synthesis as well as sonication for fibril fragmentaion which is a critical step for inducing formation of α-syn inclusions.

This updated protocol has been specified for use in vitro, and has been confirmed for use in both rat and mouse primary cultures.

[BEFORE_START]
Sonicating Fibril

Proper sonication is a key step for the fibril model to work. For all in vivo work which involved injecting fibrils into mice or rats, we use the QSonica 700 sonicator with cup horn and tube rack for 1.5 mL polypropylene tubes with a chiller at 16°C. The cup horn sonication produces short fragments which maintain their morphology for 6-8 hours (at least) and can be stored in dry ice overnight, thawed and maintained at room temperature, and therefore remain active after overnight shipments.We found that over time, the heat generated by a probe tip sonicator causes the fibrils to form amorphous aggregates (Figure 1). This is a problem because stereotaxic surgeries can take several hours and the amorphous aggregates that form while the fibrils sit on the bench causes variability and reduces the concentration of seeding competent fragments. Another advantage of using the cup horn sonicator over probe tip is that 25 μL of fibrils can be sonicated, reducing the volume needed. This is also a closed tube system which increases safety. For neuron or cell culture work in which the fibrils are added to media and then the cells immediately after sonication, a probe tip sonicator is okay. Again, this should be performed in a BSL2 hood with all proper PPE (nanoparticle respirator, goggles, gloves etc.). The volume of fibrils to be sonicated cannot be less than 100 μL.

In all cases, we wear PPE when working with fibrils. We clean any spills with 1% SDS. 


 


[GUIDELINES]
This protocol is a modification from previously published manuscripts (Patterson et al., 2019; Polinski et al., 2018; Stoyka et al., 2020; Volpicelli-Daley, Luk, & Lee, 2014).

For safe handling of fibrils please read Bousset L et al. (2016) An Efficient Procedure for Removal and Inactivation of alpha-Synuclein Assemblies from Laboratory Materials J Parkinsons Dis.6:143-51  https://pubmed.ncbi.nlm.nih.gov/26639448/

When opening tubes and pipetting, perform in a BSL2 safety hood to prevent contamination.
References: 
 

 

 

 





[STEPS]
SECTION: PFF Sonication
5. Transfer to   within cell culture hood
 

SECTION: PFF Sonication
2. Fill Qsonica water reservoir with about 900 mL. 
 

SECTION: PFF Sonication
3. Turn on sonicator and recirculating chiller. Set the temperature at 10 °C and allow to cool.

SECTION: PFF Sonication
6. Transfer tube to Qsonica tube holder for placement within the cup horn
 

 
 

SECTION: PFF Sonication
4. Thaw 30 µL aliquots of PFFs at 0.200 µg/µL   at Room temperature 
 

SECTION: PFF Sonication
1. Sonicator Info:
 

SECTION: PFF Sonication
7. Total sonication time 15 min
Pulse on 3 s and off3 s 
Temperature 10 °C 
Amplitude at 45%
Sonication output should be between 90-100kJ
 

SECTION: PFF Treatment
10. Age cultures for desired treatment length before fixation or collection.
 

SECTION: PFF Treatment
9. Add 25 µL (0.5 µg) to each well of a 24-well plate. Equal volume 1x PBS was added for control conditions.
 

SECTION: PFF Treatment
8. In culture hood, dilute PFFs to a volume of 300 µL in 1x PBS for a concentration of 0.02 µg/µL