Microglia Isolation by Immunostaining Coupled Cell Sorting: An Immunostaining Method to Isolate Microglia from Zebrafish Brain Cells Using Fluorescent Activated Cell Sorting

Overview

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.

1. 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 from cells of interest, second for the secondary antibody (1/200) to measure the non-specific binding of the secondary antibody to microglia, and third as a test (4C4 mouse monoclonal antibody (microglia specific) (1/20) + secondary antibody (1/200)).
   1. Add Low Endotoxin, Azide-Free (LEAF) at 1% to cells (all tubes) to block CD16/CD32 interactions with the Fc domain of immunoglobulins. Incubate cells for 10 min with gentle agitation every 5 min.
   2. Add the 4C4 antibody (1/20) to cells (tube 3) and incubate for 30 min with gentle agitation every 10 min.
   3. Spin tubes at 300 g for 10 min at 4 °C, then discard the supernatant.
   4. Wash once with 0.5 mL of Media A + 2% NGS, then spin tubes at 300 x g for 10 min at 4 °C.
   5. Resuspend cell pellet with 0.5 ml of Media A + 2% NGS and incubate cells with LEAF at 1% for 10 min with gentle agitation every 5 min.
   6. Add secondary antibody (1/200) to cells (tube 2 and 3). Incubate cells for 30 min with gentle agitation every 10 min and light protection.
   7. Spin tubes at 300 g for 10 min at 4 °C, then discard supernatant.
   8. Wash twice with 0.5 mL of Media A + 2% NGS, then resuspend cell pellet with 1 mL of Media A + 2% NGS.
2. Run cell suspension through a 35 µm cell strainer cap and transfer them into cold 5 mL fluorescence-activated cell sorting or FACS tubes on ice, protected from light.

2. Cell Sorting (FACS)

NOTE: Perform all steps at 4 °C.

1. Sort neurons, macrophages/microglia, and microglia using a FACS.
   NOTE: This step is usually performed by a staff member of the FACS facility and settings depend on the type of equipment used.
   1. Add DAPI at a concentration of 1 µg/mL in each FACS tube to label dead cells.
   2. Set up FACS and sort neurons, macrophages/microglia, or microglia from all brain cells. Separate cells from debris in function of their size and granularity, then gate single-cells by forward scatter and side scatter. Exclude dead cells by DAPI labeling from live cells. Identify neurons, macrophages/microglia, or microglia by their respective positive staining.
2. Collect cells in 1.5 mL tubes containing 1 mL of ice-cold Media A + 2% NGS on ice. Use different tubes for each cell type.
   1. Spin tubes at 300 x g for 10 min at 4 °C and then discard the supernatant.
   2. Wash once with 0.5 mL of Media A then discard the maximum of supernatant.

Materials

Name	Company	Catalog Number	Comments
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)
FACS sorter FACSAria II	BD	QMRI, FACS facility