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…
Divulgazioni
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 |
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Citazione di questo articolo
Magnetic Isolation of Microglia from Mouse Pup Brains. J. Vis. Exp. (Pending Publication), e21576, doi: (2023).