Jul 12, 2022
Primary astrocyte culture
- Xiqun Chen1,
- Qing Ye2
- 1Department of Neurology Massachusetts General Hospital Harvard Medical School Charlestown MA 02129 USA, Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network Chevy Chase MD 20815 USA;
- 2Department of Neurology Massachusetts General Hospital Harvard Medical School Charlestown MA 02129 USA, Department of Neurology Longhua Hospital Shanghai University of Traditional Chinese Medicine Shanghai 200032 China
Protocol Citation: Xiqun Chen, Qing Ye 2022. Primary astrocyte culture. protocols.io https://dx.doi.org/10.17504/protocols.io.b4yfqxtn
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: February 11, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 58087
Keywords: ASAPCRN
Abstract
Primary astrocytes were obtained from C57BL/6 mice embryonic day 17. The dissected cortical tissue was digested, triturated, and centrifuged. The cell pellet was resuspended in high-glucose DMEM/F12 supplemented with 10% FBS. Cells were seeded in poly-L-ornithine-coated Petri plates. Nonadherent cells were removed after 5-7 days.
Protocol materials
DMEM, high glucoseThermo Fisher ScientificCatalog #11965092
Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082147
For primary astrocyte culture - Use C57BL/6J mice at embryonic day 17
Anesthetized pregnant mice (1% sodium pentobarbital, 80mg/kg), dissect their embryos and collect the cortex.
(Separate and remove the soft membrane and blood vessels, rinse the cerebral cortex in PBS, and use the ophthalmic scissor to cut pieces of the cortex)
Collect the cortices in PBS in a 50 ml tube on ice
(The 50 ml tube contains 30 ml of PBS) On ice
Transfer the cortices to 15 ml tubes containing 1.5 ml trypsin–EDTA (0.25%) and incubate it at 37 °C for 00:15:00 Dissociate the cortices by triturating with a 10 mL serological pipette 10 – 15 times
15m
Centrifuge the dissociated cortices (1500 rpm , 00:05:00 ) and resuspend the pellet in 10ml DMEM, high glucoseThermo Fisher ScientificCatalog #11965092 medium supplemented with 10% Fetal Bovine Serum, certified, heat inactivated, United StatesThermo FisherCatalog #10082147
5m
Triturate the cell suspension 10 times with a 1ml pipette
Count the cells and plate them in a density of 50,000 cells/cm2 into PLL coated T-75 flask.
After 3 h, change the culture medium once and check the growth and survival of cells under the microscope.
Put the culture flask in a 37 °C -cell incubator for 24:00:00
1d
Change the media after 24:00:00 and then every 72:00:00 . A confluent layer of the astrocytes grows at the bottom in 5-7 days followed by a top layer of microglia.
4d
After 5-7 days, put the flask on the shaker for 2 h at 200 rpm at 37℃ (incubator). Collect and discard the supernatant (consisting of microglia and some oligodendrocytes).
Wash the adherent cells with PBS and add 0.25% trypsin at 37℃ to detach the cells.
Immediately add 10 mL DMEM medium containing 10% FBS
Centrifuge the cell suspension at 1500 rpm min for 00:05:00
Resuspend the cells in a T75 flask or 24 well plate with high glucose DMEM supplemented with 10% FBS.
5m
Identification of astrocytes by immunofluorescence staining: Use GFAP antibody (astrocytic marker) to identify the population of astroctes
Transduction with BRAF (Optional)
Astrocytes were transduced with BRAFV600E, or BRAFWT, or vector lentivirus plus 8 μg/ml polybrene for 24 h.
After transduction, the cells were cultured for 120 h in high-glucose DMEM/F12 (400ul/well) with or without FBS, and the medium as well as the cells were collected for subsequent experiments.