TITLE: PROCEDURE TO INDUCE MITOCHONDRIAL DEPOLARISATION AND LYSING OF MOUSE CORTICAL NEURONS 
AUTHORS: Odetta Antico, Miratul M. Muqit

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mutations in PINK1 cause early-onset Parkinson’s disease. PINK1 becomes stabilised and active upon mitochondrial depolarisation. This leads to phosphorylation of ubiquitin and Parkin via Serine 65 residues and a feed forward mechanism whereby PINK1 phosphorylates newly formed polyubiquitin chains, generating phospho-ubiquitin, which further promotes Parkin recruitment and activation. Once activated, Parkin ubiquitylates proteins at the outer face of the outer mitochondrial membrane (OMM) and then initiates a downstream pathway that eventually leads to mitophagy, a mitochondria-specific type of autophagy. Notably, much of previous investigation into PINK1/Parkin activity has been performed in non-neuronal human cancer cells where Parkin and/or PINK1 is over-expressed. Here we report a protocol for generation of mouse embryonic cortical neuronal cultures that produce high cell yields and can be used for studying endogenous PINK1 and Parkin signalling by biochemical methods and proteomics</div></div>

[STEPS]
?. [Mitochondrial depolarisation]
To depolarize or uncouple mitochondrial membrane potential in neurons, cultures were treated for  to  with a combination of  Antimycin A and  Oligomycin dissolved in DMSO at .
◊TIMING  - , day of experiment in this section.
10
1
37 °C
?. [Lysing of mouse neuronal cultures]
Gently aspirate the medium from wells which neurons are plated.
◊TIMING  -  in this section.
?. [Lysing of mouse neuronal cultures]
Wash twice by adding  of warmed DPBS containing protease inhibitors, phosphatase inhibitors and  Chloroacetamide.
1 mL
200
Note: Chloracetamide is added to the PBS and the lysis Buffer to inactivate deubiquitinase enzymes and enhance the detection of Ubiquitin signal.
?. [Lysing of mouse neuronal cultures]
Place the 6 well multidishes  and add  of Lysis Buffer. Carefully scrape the cells and collect the lysate in a  microcentrifuge tube.
on ice
50 µl
1.5 mL
?. [Lysing of mouse neuronal cultures]
Sonicate the cell lysate with a probe sonicator 5 seconds 20% amplitude and incubate for .
on ice
?. [Lysing of mouse neuronal cultures]
Centrifuge the cell lysate at  in a refrigerated centrifuge for . Supernatants were collected and protein concentration was determined by using the Coomassie Protein Assay.
Centrifuge: 17000 34
?. [Lysing of mouse neuronal cultures]
Cell lysates are stored at .
-80 °C
?. [Immunoblotting of mouse neuronal cultures]
of cell lysates are diluted 4X into 4X LDS loading buffer supplemented with fresh 10% 2-mercaptoethanol.
◊TIMING  -  in this section.
40 µg
?. [Immunoblotting of mouse neuronal cultures]
Samples are boiled for  at  or for  at .
97 °C
37 °C
Do not boil the sample for an extended time, ubiquitin can refold on membrane and obscure epitope
?. [Immunoblotting of mouse neuronal cultures]
Analyse samples by running on Nu-page Bis-Tris 4-12% gels for a better resolution of ubiquitin chains, at 120 V for ~.
?. [Immunoblotting of mouse neuronal cultures]
Transfer gel on PVDF membrane for phospho-ubiquitin and ubiquitin signal and nitrocellulose membrane for phospho-Parkin and Parkin signal. Transfer in Towbin buffer at 80 V for  or in cold room.
on ice
Note: Prepare only 1 membrane per transfer tank -avoid multiple membranes for transfer in same tank as this reduces ubiquitin transfer.
?. [Immunoblotting of mouse neuronal cultures]
Incubate membrane with blocking buffer 5% milk in 0.1% TBS-Tween for  at .
37 Room temperature
?. [Immunoblotting of mouse neuronal cultures]
Remove blocking buffer, if primary antibodies are in 5% BSA, rinse twice with 0.1% TBS-Tween to remove any traces of milk, add primary antibodies and incubate  at .
4 °C
Note: Prepare phospho-Ubiquitin (1:2000), Ubiquitin (1:1000), GAPDH (1:5000) and Parkin (1:1000) in 5% BSA (TBS-Tween). Prepare phosphoParkin (1:2000) in 5% milk (TBS-Tween). To avoid non-specific signal, it is recommended to preincubate phospho-Parkin antibody with a membrane for 2 days before using it.
?. [Immunoblotting of mouse neuronal cultures]
Remove primary antibody and wash 3 times with 0.1%TBS-Tween for .
?. [Immunoblotting of mouse neuronal cultures]
Add HRP-conjugate secondary antibodies for  at RT diluted 1:5000 in 1% BSA (0,1% TBS-Tween).
?. [Immunoblotting of mouse neuronal cultures]
Remove secondary antibody and wash 3 times with 0.1%TBS-Tween for .
?. [Immunoblotting of mouse neuronal cultures]
Develop signal using ECL western Blotting reagents and analysing with Chemidoc.
Note: Depending on signal, film can be best for sensitivity.