TITLE: Isolation and Culture of Mouse Cortical Neurons
AUTHORS: Ashley V Kumar, Patricia  Montilla-Perez, Francesca Telese

[DESCRIPTION]
We provide a detailed protocol to isolate and culture primary cortical neurons from the cortex of mice at embryonic day 15.5. 

[BEFORE_START]
 
Coat 10cm dishes with Poly-D-Lysine . Keep the dishes in the tissue culture incubator at 37C for 1 hour.
Rinse the dishes twice with sterile H2O.  
Leave the dishes drying in the TC incubator while isolating neurons.  


Prepare reagents (details in materials)
Complete Neuralbasal medium (NBC) 
HBSS-G
0.25% Trypsin in HBSS-G
Soybean Trypsin Inhibitor

(Optional) Filter reagents with 0.22um filters 


Tissue dissection
Before starting:
Spray down the dissection bench with 70% isopropanol. 
Prepare dissection tools
Prepare an ice bucket for chilling dissection dishes.
Place on ice 15ml tubes with 14ml HBSS-G for cortices isolated from 4 embryos 
Pre-warm NBC media and 1:1 0.25% Trypsin/ HBSS-G and warm HBSS-G at 37 ̊C in a water bath. Use 5ml of Trypsin/HBSSG for each 15ml tube and 15ml of media for 4 embryos. 
Thaw 10X DNAse 1 (1mg/ml) on ice (can remove from -20C on step 6). Dilute 1:10 in Soybean Trypsin Inhibitor medium before use (0.1mg/ml final concentration). Use 1ml of DNAse/Trypsin inhibitor medium for each 15ml tube

[GUIDELINES]
This protocol is designed to generate primary cultures of cortical neurons from the cortex of E15.5 mice.  The protocol is designed based on the dissection of brain tissues from 4 embryos, but it can be scaled up as needed.   
The protocol is structured in two sections:
1. Tissue Dissection
2. Culture plating and maintenance


The length of the protocol is ~9 days  
Please Note: Breeding and euthanasia of all animal work should be performed in accordance with an institutionally approved animal care and use protocol.

[STEPS]
SECTION: Tissue Dissection
1. Collect the embryos in a 10 cm Petri dish with ice-cold HBSS-G and cut the heads.  Keep the Petri dish on ice while dissecting. 
SECTION: Tissue Dissection
2. Using a microscope, insert the tip of tweezers into the eyes to keep the head fixed, cut up from the cranial floor to the nose, open the skull like a book, and lift the cortices with tweezers. Using a clean 10cm Petri dish with ice-cold HBSS-G for each 3-4 embryos reduces excessive contamination with blood.
SECTION: Tissue Dissection
3. Dissect the cortex by removing hindbrain and olfactory bulbs, and trim away meninges. Use tweezers to lift the meninges without puncturing the cortex tissue.
SECTION: Tissue Dissection
4. Transfer the cortex to the 15ml tube containing 14ml of HBSS-G on ice. 4 heads per 15mL tube
SECTION: Culture Plating and Maintenance
5. After dissections are complete, work inside the biosafety cabinet. Aspirate the HBSS-G, leaving as little of the dissection medium as possible without disturbing the tissue or exposing it to air. Add ~5ml/tube of warm HBSS-G/trypsin. Incubate for 20 minutes at 37C in a water bath. Invert the tube  every 5 minutes. 
Meanwhile, dilute the DNAse I 1:10 in warm trypsin-inhibitor solution.
SECTION: Culture Plating and Maintenance
6. Remove the tube containing trypsin and pieces of tissue from the water bath.  Spray it with 70% ethanol and wipe it down before moving it into the hood. Aspirate trypsin solution and add ~5ml warm HBSS-G. Let the pieces of tissue settle to the bottom of the tube for 1-2 minutes. 
SECTION: Culture Plating and Maintenance
7. Aspirate HBSS-G, leaving as little of the dissection medium as possible without disturbing the tissue or exposing it to air. Add 1ml NB/trypsin-inhibitor/DNAse I. 
SECTION: Culture Plating and Maintenance
10. Spin cells for 5 minutes at 1200 rpm at RT. Resuspend the pellet in warm culture medium (NBC). If a blood ring is present in the cell pellet, avoid carrying over the blood. Use a total volume of ~ 1ml/embryo. 
SECTION: Culture Plating and Maintenance
11. Count the live cells with trypan blue.
SECTION: Culture Plating and Maintenance
12. Plate the cells in desired culture dishes. Use ~50% of final volume of NBC for initial plating. Incubate at 37 in the incubator for 5 minutes.
SECTION: Culture Plating and Maintenance
13. Aspirate the initial culture media and replace it with fresh warm NBC (final volume). This step helps the removal of floating dead cells and debris. For example, for a 10 cm dish, use 10ml as final volume.
SECTION: Culture Plating and Maintenance
14. Change 50% of culture media every 3 days. Add AraC at a final concentration of 4uM starting from day 3. Cultures are maintained until DIV 9. We noticed that after DIV9 the viability is reduced. 
SECTION: Culture Plating and Maintenance
8. Triturate tissue pipetting up and down using a 1ml tip. Avoid introducing bubbles. Using the fewest strokes possible to dissociate the tissue is important to improve cell survival. Triturating over 15 times seems to reduce neuron survival. Let any chunk of non-dissociated tissue sink at the bottom of the tube for 1-2 minutes. 
SECTION: Culture Plating and Maintenance
9. Transfer dissociated neurons to a new 50ml tube with a nested 40um mesh pre-rinsed with NBC (~2-3mL) and leave the chunks at the bottom. After transferring neurons, do a post-rinse of the 40um mesh with NBC.