Magnetic Isolation of Microglia from Mouse Pup Brains

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Brain dissociation and magnetic microglial isolation

NOTE: All cell manipulations and resuspensions must be performed with a 1,000 µL pipette with great caution. Applying a high mechanical action may activate or kill microglia cells.

1. Transfer 12 brain pieces (~1.2 g) per dissociation tube containing dissociation mixture according to Table 1. For 24 pups, four C-Tubes will be needed (Figure 1A-....

Results

Figure 1: Schematic representation of brain dissociations and microglial cells' isolation. (A-C) After P8 mouse brain dissection and removing olfactive bulbs and cerebellum, brains were first transferred to a Petri dish with HBSS+/+, and then to dissociation tubes containing the dissociation mixture. The C-tubes were placed on the dissociator (with the heating mode), and the NTDK program was started (

Materials

Name	Company	Catalog Number	Comments
Bovine Serum Albumin	Miltenyi Biotec	130-091-376
CD11b (Microglia) MicroBeads, h, m	Miltenyi Biotec	130-093-634
D-PBS (10x)	Thermo Scientific	14200067
Falcon Cell culture 12-well plate, flat bottom + lid	Dutscher	353043
Falcon tubes 50 mL	Falcon tubes 50 mL	352098
Neural Tissue Dissociation Kit - Papain	Miltenyi Biotec	130-092-628
Nucleocounter NC-200	Chemometec
Nun EZFlip Top Conical Centrifuge Tubes	Thermo Scientific	362694
OPTILUX Petri dish - 100 x 20 mm	Dutscher	353003
gentleMACS C Tubes (4 x 25 tubes)	Miltenyi Biotec	130-096-334
gentleMACS Octo Dissociator with Heaters	Miltenyi Biotec	130-096-427
Hanks' Balanced Salt Solution (HBSS) +CaCl2 +MgCl2 10x	Thermo Scientific	14065049
Penicillin-streptomycin (10 000 U/mL)	Thermo Scientific	15140122
Macrophage-SFM serum-free medium	Thermo Scientific	12065074
MACS BSA Stock Solution	Miltenyi Biotec	130-091-376
MACS SmartStrainers (70 μm), 4 x 25 pcs	Miltenyi Biotec	130-110-916
MultiMACS Cell24 Separator	Miltenyi Biotec
Multi-24 Column Blocks	Miltenyi Biotec	130-095-691