Jul 10, 2024
Primary cortical astrocyte isolation and culture
- 1Duke University
Document Citation: Shiyi Wang 2024. Primary cortical astrocyte isolation and culture. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9dqqmg3e/v1
License: This is an open access document 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
Created: May 23, 2023
Last Modified: August 13, 2024
Document Integer ID: 82322
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Primary cortical astrocyte isolation and culture
1. P1 rat cortices from both sexes were micro-dissected, papain digested, triturated in low and high ovomucoid solutions, and resuspended in astrocyte growth media (AGM: DMEM (GIBCO 11960), 10% FBS, 10 μM, hydrocortisone, 100 U/ml Pen/Strep, 2 mM L-Glutamine, 5 μg/ml Insulin, 1 mM Na Pyruvate, 5 μg/ml N-Acetyl-L-cysteine).
2. Between 15-20 million cells were plated on 75 mm2flasks (non-ventilated cap) coated with poly-D-lysine and incubated at 37°C in 10% CO2.
3. On DIV 3, the removal of non-astrocyte cells was performed by forcefully shaking closed flasks by hand for 10-15 s until only an adherent monolayer of astrocytes remained.
4. AraC was added to the media from DIV 5 to DIV 7 to eliminate contaminating fibroblasts. On DIV 7, astrocytes were trypsinized (0.05% Trypsin-EDTA) and plated into 12-well or 6-well dishes.
5. On DIV 8, cultured rat astrocytes were transfected with shRNA and/or expression plasmids using Lipofectamine LTX with Plus Reagent (Thermo Scientific) per the manufacturer’s protocol.
6. Briefly, 1 μg (12-well) or 2 μg (6-well) total DNA was diluted in Opti-MEM containing Plus Reagent, mixed with Opti-MEM containing LTX (1:2 DNA to LTX), and incubated for 30 minutes.
7. The transfection solution was added to astrocyte cultures and incubated at 37°C for 3 hours. On DIV 10, astrocytes were trypsinized, resuspended in NGM plus, plated (20,000 cells per well) onto DIV 10 neurons, and co-cultured for 48 hours.