Magnetic Isolation of Microglia from Mouse Pup Brains

Published: August 31, 2023

Abstract

Source: Bokobza, C. et al., Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures. J. Vis. Exp. (2022)

In this video, we describe a protocol for isolating CD11b cells, including microglia, from mouse pup brains using magnetic-activated cell sorting. The brain tissues are homogenized to obtain single-cell suspensions, which are incubated with anti-CD11b antibody-coated microbeads, followed by a magnetic column-based separation to purify the CD11b+ cell population.

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. Transfer 12 brain pieces (~1.2 g) per…

Representative 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 (<strong…

開示

The authors have nothing to disclose.

Materials

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

タグ

Play Video

記事を引用
Magnetic Isolation of Microglia from Mouse Pup Brains. J. Vis. Exp. (Pending Publication), e21576, doi: (2023).

View Video