Citation
- Authors: Gutiérrez-Vázquez C. et al.
- Year: 2021
- Journal: STAR Protoc 3 101033
- Applications: in vitro / siRNA / INTERFERin
- Cell type: Mouse newborn primary astrocytes
Description: Primary mouse newborn astrocytes
Method
In this section we describe how to silence primary murine astrocytes with siRNAs using INTERFERin (Polyplus-transfection):
- Mix INTERFERin reagent with the desired siRNAs in Opti-MEM following Table 3.
- Incubate the mix 10 min at room temperature.
- Change the media of the astrocytes to fresh new media (Complete DMEM or N1-DMEM) following volumes on Table 3.
- Add the mix of INTERFERin, siRNAs and Opti-MEM dropwise to the astrocyte’s wells and incubate at 37°C.
Abstract
Robust protocols are required to investigate in vitro the molecular mechanisms that control astrocyte metabolism and pro-inflammatory activities. In the present protocol, we describe step by step the isolation and culture of primary murine astrocytes from neonatal brains, followed by their genetic manipulation with siRNA. We further describe cytokine activation of the cultured astrocytes for the analysis of their pro-inflammatory responses, and the oxygen consumption analysis to assess their metabolic function. For complete details on the use and execution of this protocol, please refer to Chao et al. (2019), Clark et al. (2021), and Rothhammer et al. (2018).