Feb 17, 2025
Version 1
Primary Neuron Extraction V.1
- 1Caltech
Protocol Citation: Yujie Fan, Chelsie Steele 2025. Primary Neuron Extraction . protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo98ozv4o/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: January 15, 2025
Last Modified: February 17, 2025
Protocol Integer ID: 118316
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Before staring the surgery:
Before staring the surgery:
30m
30m
Turn Dry Sterilizer on 30 min to heat, once heated put in scissors and 4 pair needle nose forceps
30m
Make sure there is enough plating medium. Plating media consists of - 50 ml brain phys plating media+ 125 ul glutamax + 500 ul Penicillin-Streptomycin + 92.5 of l-glutamine + 1 ml sm1
Coat however many plates you want to use with Poly-L-Ornithine Coating and incubate for 1 h at 37 °C.
Make 2 15 ml tubes of HBSS 1x – our current stock is 10x. Dilute by adding 1 ml of HBSS 10x to 9 ml distilled water
Prep Papain and HBSS 1x mixture in 1.5 ml tubes. 1 1.5 ml tube for 3 cortices When prepping papain be sure to check the bottle as the amount added to HBSS 1x depends on the units/ml . Current bottle is 24 mg/ml and 16 units/mg (16 * 24 = 384 units/ml). Want 15/384 = 0.04 so 40 ug papain per 1 ml HBSS 1x
Prep/check that there is enough HBSS 1x + 10% FBS. This is the "stopping medium" Need at least 3 ml per each 15 ml tube of 3 cortices
Collect tools: scissors and 4 pair needle nose forceps
Sterilize 1 min and let cool
Dissection in fume hood
Dissection in fume hood
5m
5m
Euthanize female mouse at day 17 of her pregnancy with CO2 bubble at 1 for until no breath ~5 min
5m
Place AT cooling Core in container
Collect 3 sterile 100mm plates, fill one with HBSS 1X from fridge and place on the cooling core
Prepare slide box with paper towels, place dead female on her back on the towels and spray very well with 70% Ethanol
Lift skin with sterile forceps, use scissors to cut low and upward to expose embryos, lift sac and carefully snip out uterus trying not to nick any organs. (5 embryos are enough for 2 24 well plates)
Place embryos still in uterus in cooled HBSS 1x media
Put female in carcass bag and in freezer
Using 2 forceps remove embryos from sac and place in dish with HBSS 1X
Position embryo back up and pin behind the head with forceps
Peel back skin and skull, place tip of forceps under cerebellum, lift up and forward to remove the brain from the skull
Move embryo out of solution into the dish with HBSS 1x
Repeat with all embryos
Hold down cerebellum with forceps. Lift and snip off, each cortical lobe, with forceps; the hippocampus comes off with the cortex. Move the rest of the brain to the HBSS 1x and repeat with the rest
Take off the meninges from the cortex by stabilizing the cortex with tip of forceps and gently, tearing off the meninges with the other forceps
Flip over the cortex and take out the hippocampus (Watch video on how to identify the hippocampus)
Quarter each hippocampus and transfer sections to a 15ml falcon tube using a cut 1ml pipette tip.
Remove as much HBSS from the tube as possible then go to the TC
Tissue Culture room
Tissue Culture room
20m
20m
Add 1ml papain/HBSS mixture to each 15 ml falcon tube
Put 15 ml falcon tube in water bath for a total of 15 min, stopping to shake the tubes at 7.5 minutes.
15m
Remove the digested hippocampal sections from the incubator and let pieces settle on the bottom before removing the solution with a bulb and glass pipette leaving 1ml of solution
Add 5ml HBSS 1X, 10% FBS to rinse the pieces once, wait 20 seconds then remove most of the solution. Repeat wash step once more
Add 3ml HBSS 1X and start dissociation by pipetting gently 10 times with the glass pipette. The supernatant will contain the dissociated neurons
Let the bigger pieces settle and transfer the supernatant to a new 15ml Falcon tube. Alternatively, transfer the big pieces to a new tube
Last dissociation, add 2ml HBSS 1X to hippocampal pieces and pipette up and down 5 times and transfer supernatant to dissociated neuron tube, roughly 6ml total. Check for large pieces and try to remove them.
Centrifuge 5 min at 1000rpm at RT. Warm complete media in 37oC water bath while spinning.
5m
Remove the supernatant from pelleted cells. Add 8ml warmed complete brain phys media.
Gently resuspend with pipette, up and down 6 times.
Count cells and plate accordingly
Perform a half to full medium change on cell culture dishes at 3 day intervals
Protocol references
We follow the guidelines given in the paper "Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression" doi:10.3791/54981