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

[STEPS]
?. [Coating of multiwell dishes with poly-L-lysine]
In a sterile laminar hood, prepare poly-L-lysine  in sterile from stock solution.
◊TIMING  , 1 d before culture in this section.
?. [Coating of multiwell dishes with poly-L-lysine]
Add enough poly-L-lysine solution to cover the bottom of the well, ensure that the volume of poly-L-lysine covers the bottom of the dish entirely, usually  for 1 single well.
1 mL
?. [Coating of multiwell dishes with poly-L-lysine]
Wrap the dish in aluminum foil to prevent evaporation and leave it  at.
0 Room temperature
It is important to make sure that the poly-L-lysine does not dry out during incubation. If you are working in the laminar hood, be sure to turn the blower off during the incubation
?. [Washing of dishes after coating]
Aspirate the poly-L-lysine carefully.
◊TIMING  , day of culture in this section.
?. [Washing of dishes after coating]
Add  of sterile water into each well and aspirate; repeat this step three times.
1 mL
?. [Washing of dishes after coating]
Aspirate water and leave dishes under the hood until completely dry.
?. [Dissection of cortex from E16.5 mouse embryos]
Use sterilized instruments by autoclave or washing them with 70% (vol/vol) ethanol.
◊TIMING  ,  –  for each embryo in this section.
Dry thoroughly if ethanol is used.
?. [Dissection of cortex from E16.5 mouse embryos]
Prepare 60-mm dishes with dissection medium. If you are culturing from individual embryos, prepare  tubes with  of dissection medium. If you are culturing pooled embryos, prepare  tubes with  of dissection medium.
15 mL
1 mL
15 mL
5 mL
?. [Dissection of cortex from E16.5 mouse embryos]
Euthanize the embryos pup by decapitation and separate the head from the body.
?. [Dissection of cortex from E16.5 mouse embryos]
Place the head on a dish with dissection medium and hold down the sides with forceps.
?. [Dissection of cortex from E16.5 mouse embryos]
Under a dissecting microscope, dissect the skin on the top of the head and hold down the skin on either side with the forceps.
?. [Dissection of cortex from E16.5 mouse embryos]
Cut open the skull by making an incision at the base of the brain. Separate the two halves of the skull and remove carefully.
Take care to not cut through the brain tissue when removing the skull bone
?. [Dissection of cortex from E16.5 mouse embryos]
By using forceps, pinch off the brain from the base and peel off the meninges carefully, ideally as a single piece. Check for the remaining pieces of meninges and remove them completely.
The meninges can be sticky and difficult to remove, it is important to ensure that the meninges are completely removed, so that they do not contribute any non-neuronal cells to the culture.
?. [Dissection of cortex from E16.5 mouse embryos]
Separate the two halves of the brain by making a sagittal cut along the midline.
?. [Dissection of cortex from E16.5 mouse embryos]
Orient the tissue so that the hippocampus is on the top. The hippocampus can be identified by its C-shaped structure and opacity, which differ from the neighboring cortical tissue. Cut away hippocampus and flat the cortex to expose the striatum. Using an angled forceps scoop out the striatum and cortex can also be dissected out and processed for cortical cultures.
It is important to ensure that the hippocampus and striatum are completely removed, so that they do not affect cortical distribution of neurons and contribute to an increase of glia cells.
?. [Dissection of cortex from E16.5 mouse embryos]
To culture neurons from individual embryo, put each brain in a separate  tube containing  of dissection medium. For pooled cultures, put cortices from two embryos into one  tube containing  of dissection medium.
15 mL
1 mL
15 mL
5 mL
Ensure that the cortices are submerged in the medium and do not let them dry out at any point. It is important that the dissection be done as quickly as possible to ensure cell viability and health.
?. [Dissection of cortex from E16.5 mouse embryos]
For individual cultures, collect a piece of tail for genotyping.
?. [Cell dissociation and plating]
Prepare digestion medium by adding  of DNase I (stock solution  ) to  of Trypsin 0,05%.
◊TIMING ~ -   in this section.
125 µl
10 mL
?. [Cell dissociation and plating]
Add 1:1 digestion medium to dissection medium containing the cortices, for individual mouse culture add  of digestion media, for pooled mouse culture add  of digestion media.
1 mL
5 mL
Note. It is common to use HBSS Ca2+ and Mg2+ free buffer. In our hands, we have noticed that using HBSS plus Ca2+ and Mg2+ ensure a milder but optimal concentration of trypsin that allows to reduce excessive number of dead cells and higher yield of cells. At the same time, it stimulates DNase I activity.
?. [Cell dissociation and plating]
Incubate at  in a water bath for .
37 °C
It is important to ensure that this incubation does not proceed for longer than
?. [Cell dissociation and plating]
Inactivate trypsin digestion by adding dissociation medium,  for individual mouse culture or  for pooled mouse culture.
2 mL
5 mL
?. [Cell dissociation and plating]
Centrifuge at  for .
Centrifuge: 1200 33
?. [Cell dissociation and plating]
Resuspend cortices in  (for individual embryos) or  (for pooled embryos) of dissociation medium.
2 mL
4 mL
?. [Cell dissociation and plating]
Carefully triturate the tissue 20 times to dissociate the cells gently and obtain a homogenous cell suspension, by using a p1000 pipette.
The trituration of cells should be done slowly and carefully to minimize damage to cells. It is best to avoid any bubbling during the procedure.
?. [Cell dissociation and plating]
Remove any chunks of tissue by using a cell strainer and distribute drop by drop the cell suspension.
?. [Cell dissociation and plating]
Centrifuge at  for  to pellet down the cells and resuspend them in the culturing medium.
Centrifuge: 700 33
Note: It is important to remove FBS from the medium to reduce the proliferation of glial cells.
?. [Cell dissociation and plating]
Resuspend the dissociated cells in  (for individual embryos) or  (for pooled embryos) of culturing medium.
2 mL
4 mL
?. [Cell dissociation and plating]
Take a  aliquot, add 1:1 ratio Trypan Blu and determine the density of cells and cell viability to the cell counter. Typical yields are ~2 x106/mL and viability >80 %.
15 µl
?. [Cell dissociation and plating]
5.0 × 105  cells/well plates are plated out on 6-well multidishes, containing  of pre-warmed culturing media.
2 mL
?. [Maintenance of neurons]
Every 5 days aspirate 1/3 of the media from each well and replace it with fresh culturing medium warmed to . These neurons can be maintained in culture for up to DIV 28 and be used anytime during this period (DIV0-28). The culture can be used for examining PINK1 activation at 21DIV, since at this stage they express functional activity of Parkin and PINK1.
◊TIMING   Up to   in this section.
37 °C
It is important to not replace the entire medium, neurons secrete factors that promote growth and survival.