Magnetic Isolation of Microglia from Mouse Pup Brains

In This Article

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 dissociation tube containing dissociation mixture according to Table 1. For 24 pups, four C-Tubes will be needed (Figure 1A-B).
Place C-Tubes on the dissociator (with the heating mode). Start the optimized NTDK program in the equipment according to the manufacturer's instructions (Figure 1D).
Centrifuge at 300 x g for 20 s (at 4 °C) to collect all the cells. Complete the mechanical dissociation by pipetting three times up and down with a 1,000 µL pipette (Figure 1E).
Transfer the cells to four 15 mL tubes + strainers. Rinse the strainers with 10 mL of 1x Hanks' Balanced Salt Solution (HBSS) with Ca²⁺ and Mg²⁺ (HBSS+/+) (Figure 1F).
Centrifuge at 300 x g for 10 min (at 4 °C) and remove the supernatant. Carefully resuspend the pellet with 10 mL of HBSS+/+ (Figure 1G).
Centrifuge at 300 x g for 10 min (at 4 °C) and remove the supernatant. Carefully resuspend the pellet with 6 mL of sorting buffer (1x PBS + 0.5% BSA) (Figure 1H).
Centrifuge at 300 x g for 10 min (at 4 °C) and remove the supernatant. Add 200 µL of CD11b-microbead solution per tube and resuspend carefully (Figure 1I).
Incubate the tubes for 15-20 min at 4 °C. Carefully resuspend the pellet with 6 mL of sorting buffer (Figure 1I-J).
Centrifuge at 300 x g for 10 min (at 4 °C) and remove the supernatant. Carefully resuspend the pellet with 8 mL of sorting buffer (Figure 1K).
Follow the POSSEL program on the separator (see Table of Materials) to prepare eight columns. Transfer cells 1 mL by 1 mL on the column. Wait for all the cells to pass through before adding another mL. Elute CD11b+ cells on a sterile elution plate with 1 mL of sorting buffer (Figure 1L).
Pool CD11b+ cells in a new 50 mL tube (Figure 1M).
Centrifuge at 300 x g for 10 min (at 4 °C) and remove the supernatant. Carefully resuspend the pellet with 10 mL of cold microglia medium (macrophage serum-free culture medium (SFM) + 1% of Penicillin-Streptomycin (P/S)) (Figure 1N).
Count the CD11b+ cells. At P8, one should obtain ~650,000 cells per brain.
NOTE: In the present protocol, the cells were counted using an automated cell counter (see Table of Materials).
Resuspend the cells in cold microglia medium to a final concentration of 650,000-700,000 cells/mL and dispense in cell culture plates.
NOTE: The 6-well plates are for Western Blot (2 mL per well); the 12-well plates are for RT-qPCR analysis (1 mL per well), and the 96-well plates are for phagocytic assay (250 µL per well).

Table 1: Preparation of the dissociation mixture.

Solution	For one C-Tube (μL)	For four C-tubes (μL)
Buffer X	2850	11400
Enzyme P (Papain)	75	300
Enzyme A (DNAse)	15	60
Buffer Y	30	120
Total	2970	11880

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Results

Brain cell isolation process; mouse brain dissection, centrifugation steps; diagram for research use.
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 con...