Jul 10, 2024
Primary cortical neuron isolation and culture
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Primary cortical neuron isolation and culture. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbz57ygpk/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: July 10, 2024
Last Modified: July 10, 2024
Protocol Integer ID: 103181
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Primary cortical neuron isolation and culture
**Dissection and Digestion** - Micro-dissect cortices from P1 rat pups of both sexes (Sprague Dawley, Charles River Laboratories, SD-001). - Digest cortices in papain (~7.5 units/ml) at 33°C for 45 minutes.
**Cell Preparation** - Triturate the digested tissue in low and high ovomucoid solutions. - Resuspend cells in panning buffer (DPBS (GIBCO 14287) supplemented with BSA and insulin). - Pass cells through a 20 μm mesh filter (Elko Filtering 03-20/14).
**Negative Panning** - Incubate filtered cells on negative panning dishes coated with: - Bandeiraea Simplicifolia Lectin 1 (x2) - Goat anti-mouse IgG+IgM (H+L) (Jackson ImmunoResearch 115-005-044) - Goat anti-rat IgG+IgM (H+L) (Jackson ImmunoResearch 112-005-044) antibodies.
**Positive Panning** - Incubate cells on positive panning dishes coated with mouse anti-L1 (ASCS4, Developmental Studies Hybridoma Bank, Univ. Iowa) to bind cortical neurons. - Collect adherent neurons by forceful pipetting with a P1000 pipette.
**Neuron Isolation and Plating** - Pellet isolated neurons (11 minutes at 200 g). - Resuspend neurons in serum-free neuron growth media (NGM): - Neurobasal, B27 supplement, 2 mM L-Glutamine, 100 U/ml Pen/Strep, 1 mM sodium pyruvate, 4.2 μg/ml Forskolin, 50 ng/mL BDNF, and 10 ng/mL CNTF. - Plate 70,000 neurons onto 12 mm glass coverslips coated with 10 μg/ml poly-D-lysine (PDL, Sigma P6407) and 2 μg/ml laminin. - Incubate at 37°C in 10% CO2.
**Media Changes and Maintenance** - On day in-vitro (DIV) 2: - Replace half of the media with NGM Plus (Neurobasal Plus, B27 Plus, 100 U/mL Pen/Strep, 1 mM sodium pyruvate, 4.2 μg/ml Forskolin, 50 ng/ml BDNF, and 10 ng/ml CNTF). - Add AraC (10 μM) to stop the growth of proliferating contaminating cells. - On DIV 3: - Replace all the media with NGM Plus. - On DIV 6 and DIV 9: - Replace half of the media with NGM Plus.
**Lentivirus Infection Protocol** - On DIV 2: - Add 100 μl of supernatant containing lentivirus plus polybrene (1 μg/ml) to the AraC NGM mixture. - On DIV 3: - Completely wash out the lentivirus and replace with NGM Plus containing 100 ng/ml BDNF.