Microglia Isolation by Immunostaining Coupled Cell Sorting: An Immunostaining Method to Isolate Microglia from Zebrafish Brain Cells Using Fluorescent Activated Cell Sorting
In this video, we isolate zebrafish microglial cells via immunostaining-coupled cell sorting. The isolated microglia can be used in downstream experiments to understand their role in brain pathologies.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Microglia Immunostaining NOTE: All steps are performed at 4 °C. Resuspend the cell pellet obtained from brain culture cells with 0.3 mL of Media A + 2% normal goat serum or NGS. Split them in 3 x 1.5 mL tubes: one for unstained cells to measure auto-fluorescence fro…
Divulgazioni
The authors have nothing to disclose.
Materials
Hepes
Gibco
15630-056
D-Glucose
Sigma
G8644-100ML
HBSS 1X
Gibco
14170-088
Normal goat serum (NGS)
Cell Signalling
5425S
35 μm cell strainer cap
BD
352235
FACS tubes
BD
352063
Low Endotoxin, Azide-Free (LEAF)
Biolegend
101321
Alexa Fluor 647 Goat Anti-Mouse IgG (H+L)
Life Technologies
A11008
Anti-4C4
Courtesy of Catherina Becker (University of Edinburgh)
Microglia Isolation by Immunostaining Coupled Cell Sorting: An Immunostaining Method to Isolate Microglia from Zebrafish Brain Cells Using Fluorescent Activated Cell Sorting. J. Vis. Exp. (Pending Publication), e20638, doi: (2023).