Jul 13, 2022
Primary microglial 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 microglial culture. protocols.io https://dx.doi.org/10.17504/protocols.io.b4ubqwsn
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 09, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 57955
Keywords: ASAPCRN, primary microglial culture mixed glial cell, primary microglial culture, mixed glial cell culture, microglial cell, mixed glial cell, mechanical shaking of the mixed glial cell culture, glial cell
Abstract
Mixed glial cells were obtained from C57BL/6 mice embryonic day 17. Cells were cultured in high-glucose DMEM/F12 supplemented with 10% FBS in humidified air containing 5% CO2 at 37°C. The culture medium was replaced with fresh medium 24 h after the initial preparation and every 3 days thereafter. After 1 week, microglial cells were obtained by mechanical shaking of the mixed glial cell cultures for 1 h. Cells were routinely monitored for purity by ionized calcium-binding adaptor molecule 1 (Iba1) staining and the population of Iba1+ cells was >95%
Protocol materials
DMEM, high glucoseThermo Fisher ScientificCatalog #11965092
Fetal Bovine Serum (FBS)ATCCCatalog #30-2020
Troubleshooting
Primary microglial 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 (400 x g , 00:05:00 ). Aspirate the media and resuspend the pellet in 5 ml DMEM, high glucoseThermo Fisher ScientificCatalog #11965092
5m
Count the cells and plate them in a density of 50,000 cells/cm2 into PLL coated T-75 flask. Makeup the volume to 15 ml with the DMEM, high glucoseThermo Fisher ScientificCatalog #11965092
Change the culture medium the next day followed by the addition of a fresh culture medium every 168:00:00 till 14 days of culture
1w
Put the flask on a shaker for 06:00:00 at 37 °C (The microglia grow as a monolayer on the top)
Collect the detached cells, centrifuge (400 x g, Room temperature , 00:05:00 ) and resuspended in 4 mL of DMEM, high glucoseThermo Fisher ScientificCatalog #11965092
Count the cells and plate at a density of 50,000 cells/cm2 in a 24 well plate
Incubate them at 37 °C in 5% CO2. After 02:00:00 microglia usually attaches to the bottom of the plate.
The purity of the cells was monitored by staining with ionized calcium-binding adaptor molecule 1 (Iba1) antibody
(Microglia obtained by this method have a purity of 90%-95% and can be identified by immunofluorescence staining.
8h 5m
Transduction with BRAF (Optional)
The purified microglial cells were seeded at a density of 80% and transduced with BRAFV600E, or BRAFWT, or vector lentivirus with 8 μg/ml polybrene (Sigma–Aldrich, USA) for 24:00:00 .
1d
After transduction, the cells were cultured in DMEM, high glucoseThermo Fisher ScientificCatalog #11965092 with or without Fetal Bovine Serum (FBS)ATCCCatalog #30-2020 for 120:00:00 , and the medium, as well as the cells, were collected for subsequent experiments.
5d