Jul 10, 2024
Rat astrocyte isolation and culture
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Rat astrocyte isolation and culture. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjnpxpgk5/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: 103187
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Rat astrocyte isolation and culture
**Cortex Dissection and Digestion** - Micro-dissect P1 rat cortices from both sexes. - Digest cortices in papain solution.
**Trituration and Resuspension** - Triturate tissue in low and high ovomucoid solutions. - Resuspend cells 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
**Cell Plating and Incubation** - Plate 15-20 million cells on 75 mm² flasks (non-ventilated cap) coated with poly-D-lysine. - Incubate at 37°C in 10% CO2.
**Non-Astrocyte Cell Removal (DIV 3)** - Forcefully shake closed flasks by hand for 10-15 seconds to remove non-astrocyte cells. - Ensure only an adherent monolayer of astrocytes remains.
**AraC Treatment (DIV 5 to DIV 7)** - Add AraC to the media to eliminate contaminating fibroblasts.
**Astrocyte Passage and Plating (DIV 7)** - Trypsinize astrocytes with 0.05% Trypsin-EDTA. - Plate cells into 12-well or 6-well dishes.
**Transfection of Astrocytes (DIV 8)** - Transfect with shRNA and/or expression plasmids using Lipofectamine LTX with Plus Reagent (Thermo Scientific). - Dilute 1 μg (12-well) or 2 μg (6-well) total DNA in Opti-MEM with Plus Reagent. - Mix with Opti-MEM containing LTX (1:2 DNA to LTX ratio). - Incubate for 30 minutes at room temperature. - Add transfection solution to astrocyte cultures and incubate at 37°C for 3 hours.
**Co-Culture with Neurons (DIV 10)** - Trypsinize astrocytes on DIV 10. - Resuspend in NGM Plus. - Plate 20,000 cells per well onto DIV 10 neurons. - Co-culture for 48 hours.