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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Abstract

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Introduction

Microglia are the central nervous system resident macrophage-like cells derived from erythropoietic precursors of the yolk sac that migrate to the neuroepithelium during early embryonic development1. Apart from their immunity functions, they also play a significant role during neurodevelopment, particularly for synaptogenesis, neuronal homeostasis, and myelination2. In adulthood, microglia develop long cellular processes to scan the environment continuously. In case of homeostasis ruptures such as brain injury or brain disease, microglia can change their morphological appearance to adopt an amoeboid shape, migrate to the injured area, increase and release many cytoprotective or cytotoxic factors. Microglia have heterogeneous activation states depending on their developmental stage and the type of injury sustained3,4,5. In this study, these activation states are broadly classified into three different phenotypes: pro-inflammatory/phagocytic, anti-inflammatory, and immuno-regulatory, keeping in mind that in reality, the situation is likely to be more complex6.

Studying in vivo microglial activation and screening for neuroprotective strategies at early stages of brain development can be challenging due to (1) the fragility of animals before weaning and (2) the low number of microglial cells. Therefore in vitro studies on microglia are widely used for toxicity7,8,9, neuroprotective strategies5,10,11,12,13,14, and co-cultures15,16,17,18,19,20,21. In vitro studies can use either microglial cell lines, stem-cell-derived microglia, or primary microglia culture. All these approaches have advantages and disadvantages, and the choice depends on the initial biological question. The benefits of using primary microglia cultures are the homogenous genetic background, pathogen-free history, and control of the time when the microglia are stimulated after animal death22.

Over the years, different methods (flow cytometry, shaking, or magnetic labeling) were developed for culturing primary microglia from rodents, both neonate and adult23,24,25,26,27,28,29. In the present work, microglia isolation from mouse neonate pups is performed using previously described magnetic-activated cell sorting technology using microbead-coated anti-mouse CD11b25,27,29. CD11b is an integrin-receptor expressed at the surface of myeloid cells, including microglia. When there is no inflammatory challenge within the brain, almost all CD11b+ cells are microglia30. Compared to other previously published methods23,24,25,26,27,28,29, the present protocol balances immediate ex vivo microglial activation analyses and common in vitro primary microglial culture. Thus, microglia are (1) isolated at postnatal day (P)8 without myelin removal, (2) cultured without serum, and (3) exposed either to siRNA, miRNA, pharmacological compound, and/or inflammatory stimuli only 48 h after brain isolation. Each of these three aspects makes the current protocol relevant and rapid. First of all, the use of pediatric microglia allows obtaining dynamic and reactive viable cells in culture without requiring an additional demyelination step that could potentially modify microglial reactivity in vitro. The present protocol aims to get as close as possible to the physiological environment of microglia. Indeed, microglia never encounter serum, and this protocol does not require the use of serum either. Moreover, exposing microglia as early as 48 h after culture prevents them from losing their physiological faculties.

Protocol

The protocol was approved, and all the animals were handled according to the institutional guidelines of Institut National de la Santé et de la Recherche Scientifique (Inserm, France). Magnetic isolation of microglia from the brains of 24 OF1 mouse pups (both male and female) at P8, divided into 6-well, 12-well, or 96-well plates, are presented. The experimental work was performed under a hood to maintain sterile conditions.

1. Preparation of sterile solutions for isolation and cell culture

  1. Prepare 50 mL of 1x Hanks' Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ (HBSS-/-) from the commercially available 10x solution (see Table of Materials).
  2. Prepare dissociation mixture according to the composition provided in Table 1 using a commercially available tissue dissociation kit (see Table of Materials).
  3. Prepare 200 mL of 1x HBSS with Ca2+ and Mg2+ (HBSS+/+) from the commercially available 10x solution.
  4. Prepare 200 mL of 1x PBS + 0.5% BSA (referred to as cell sorting buffer).
  5. Prepare CD11b-microbeads according to Table 2.
  6. Prepare 500 mL of macrophage serum-free culture medium (SFM) + 1% of Penicillin-Streptomycin (P/S). Make aliquots of 50 mL tubes and store them at 4 °C. This is referred to as the microglia medium later in the text.
    NOTE: All isolation solutions must be freshly prepared under sterile conditions on experimentation day and kept on ice. Microglia cell culture medium can be prepared, aliquoted in 50 mL tubes, and kept at 4 °C for future use. Filtration is not needed.

2. Brain dissection

  1. Decapitate the pup's head using large scissors without previous general anesthesia.
  2. Cut the skin from the neck to the nose following the sagittal suture (15-20 mm) with small scissors (Figure 1A-C).
  3. Insert a small scissor's tips within the Foramen magnum parallel to the skull. Cut from each side carefully to the eyes (Figure 1D,E).
  4. Cut between the eyes with small scissors to detach the skull and brain from the head (Figure 1F).
  5. With two forceps, grab the skull close to the olfactory bulbs and tear the skull carefully, taking care not to damage the underlying brain (Figure 1G-I).
  6. Remove cerebellum and olfactive bulb with a razor blade and cut the brain into two pieces (Figure 1J).
  7. Place the brain pieces in a Petri dish containing 30-40 mL of HBSS-/- (Figure 1K).

3. 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 were needed (Figure 2A-B).
  2. Place C-Tubes on the dissociator (with the heating mode). Start the optimized NTDK program in the equipment according to the manufacturer's instruction (Figure 2D).
  3. 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 2E).
  4. Transfer the cells to four 15 mL tubes + strainers. Rinse the strainers with 10 mL of HBSS+/+ (Figure 2F).
  5. 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 2G).
  6. 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 (step 1.4) (Figure 2H).
  7. Centrifuge at 300 x g for 10 min (at 4 °C) and remove the supernatant. Add 200 µL of CD11b-microbead solution (step 1.5) per tube and resuspend carefully (Figure 2I).
  8. Incubate the tubes for 15-20 min at 4 °C. Carefully resuspend the pellet with 6 mL of sorting buffer (Figure 2I-J).
  9. 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 2K).
  10. 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 sterile elution plate with 1 mL of sorting buffer (Figure 2L).
  11. Pool CD11b+ cells in a new 50 mL tube (Figure 2M).
  12. 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 (step 1.6) (Figure 2N).
  13. 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).
  14. 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); all three were used for this work. However, in this manuscript, the results from the Western Blot analysis are not shown, but it is possible to perform with this protocol.
  15. Place the plates at 37 °C with 5% CO2 overnight. Change the medium carefully with a 1,000 µL pipette with pre-heated microglia medium.
  16. Place the plates at 37 °C with 5% CO2 overnight before stimulation.
    NOTE: Isolating microglia from more than 36 pups and after P9 are not recommended. This will increase the risk of contamination and accumulation of cell debris to activate microglia.

4. Cell stimulations

  1. Prepare stimulation reagents according to Table 3 using the commercially available reagents (see Table of Materials).
  2. Add the appropriate volume in each stimulated well.
    NOTE: The appropriate volume depends on the concentration of the stimulus and the size of the well.
  3. Place the plates at 37 °C with 5% CO2until the end of stimulation at 6 h, 24 h, or 48 h.
  4. For the Western Blot analysis, aspirate the supernatant and add 50 µL of protein lysis buffer (see Table of Materials) with a pipette. Using tips, scratch the bottom of the plate to detach the lysed cells and transfer them to 1.5 mL tubes. Store at -80 °C.
    NOTE: The culture plates can be stored for years at -80 °C if the supernatant is aspirated correctly.
  5. For RT-qPCR analysis6,31, aspirate the supernatant and store the culture plates directly at -80 °C until mRNA extraction (step 5).
  6. For the phagocytic assay, please see step 6.

5. mRNA extraction and RT-qPCR analysis

  1. Perform RNA extraction, RT-PCR, and RT-qPCR following the manufacturer's protocol (see Table of Materials). Refer to Table 4 for the primer sequences5,6,31.
  2. Perform RT-qPCR analysis by following the previously published report5.

6. Phagocytic assay

  1. Stimulate the cells and perform phagocytic assay during the last 3 h of the stimulation. For example, for stimulation of 6 h, start the phagocytic assay after 3 h of stimulation; and for stimulation of 12 h, start the phagocytic assay 9 h after the beginning of the stimulation.
  2. Calculate the number of beads needed to prepare considering the ratio (1 cell: 50 beads). For one well of the 96-well plate, 8.1 x 106 beads are required. Prepare the beads mixture according to Table 5.
    NOTE: Carefully vortex the beads stock vial before adding to the PBS/FBS mixture.
  3. Incubate for 1 h in a water bath at 37 °C. Vortex every 10 min.
  4. Calculate the solution volume to add to each well. Add the solution and incubate for 3 h.
  5. Rinse the wells three times with 1x PBS. Read the fluorescence emission at 550 nm (Cy3 emission wavelength).

7. Purity quality control

  1. Evaluate the purity of CD11b magnetic isolation by flow cytometry (FACS) (before and after cell sorting), and then perform the RT-qPCR.
    1. Count the cells and resuspend in FACS buffer (PBS + 2 mM of EDTA + 0.5% of Bovine Serum Albumin) in order to obtain a dilution of 10 x 106 cells/mL after step 3.5 and step 3.14.
    2. After 15 min of the conventional Fc blocking32,33, incubate the cells for 15 min with viability probe (FVS780) and fluorophore-conjugated antibodies against mouse CD45, CD11b, CX3CR1, ACSA-2, O4 or their corresponding control isotypes32,33 at the concentration recommended by the manufacturers (see Table of Materials).
    3. Wash the cells with FACS buffer, fix, and permeabilize with a commercially available permeabilization kit (see Table of Materials).
    4. Perform Fc blocking again on the permeabilized cells, and then incubate with fluorophore-conjugated antibody against NeuN (see Table of Materials) or its control isotype for 15 min.
    5. Perform FACS analysis after washing with FACS buffer.
    6. After excluding the doublets and the dead cells based on morphological parameters and FVS780 staining, respectively, select the gating strategy for the surface expression of CX3CR1 (microglia), ACSA-2 (astrocytes), O4 (oligodendrocytes), and intracellular presence of NeuN (neurons) for the analysis of the percentage of positive cells.
  2. Following step 5, extract mRNA and perform RT-qPCR to quantify Itgam (CD11b), Cx3cr1, Olig2, Synaptophysin, and Gfap mRNA. Normalize Cq using Rpl13a mRNA as a reporter, and perform a relative expression to Itgam mRNA.

Results

Microglia is the CNS resident macrophage that gets activated when exposed to environmental challenges (trauma, toxic molecules, inflammation )4,5,6,34 (Figure 3A). In vitro studies on microglia are commonly used to evaluate cell-autonomous mechanisms related to those environmental challenges and characterize activation state after pharmacological or g...

Discussion

The current work presents a primary microglial cell culture using magnetically sorted CD11b+ cells. In addition to the microglial functional evaluation (RT-qPCR and phagocytic assays), microglial culture purity was also determined.

Classical microglia cell cultures are commonly generated from P1 or P2 rodent neonate brain and co-culture with astrocytes for at least 10 days. Microglia are then separated mechanically using an orbital shaker. The method to isolate and culture microglia in vit...

Disclosures

The authors declare no conflicts of interest.

Acknowledgements

Figures were created using BioRender. Research is funded by Inserm, Université de Paris, Horizon 2020 (PREMSTEM-874721), Fondation de France, Fondation ARSEP, Fondation pour la Recherche sur le Cerveau, Fondation Grace de Monaco, and an additional grant from Investissement d'Avenir -ANR-11-INBS-0011-NeurATRIS and Investissement d'Avenir -ANR-17-EURE-001-EUR G.E.N.E.

Materials

NameCompanyCatalog NumberComments
Anti mouse ACSA-2 PE Vio 615Miltenyi Biotec130-116-246
Anti mouse CD11b BV421Sony Biotechnology1106255
Anti mouse CD45 BV510Sony Biotechnology1115690
Anti mouse CX3CR1 PE Cy7Sony Biotechnology1345075
Anti mouse NeuN PEMilli-MarkFCMAB317PE
anti mouse O4 Vio Bright B515Miltenyi Biotec130-120-016
BD Cytofix/Cytoperm permeabilization kitBD Biosciences554655
Bovine Serum AlbuminMiltenyi Biotec130-091-376
CD11b (Microglia) MicroBeads, h, mMiltenyi Biotec130-093-634
Confocal microscopeLeica TCS SP8
D-PBS (10x)Thermo Scientific14200067
EDTASigma-AldrichE1644
Falcon Cell culture 12-well plate, flat bottom + lidDutscher353043
Falcon Cell culture 96-well plate, flat bottom + lidDutscher353072
Falcon tubes 50 mLDutscher352098
Fc blocking reagent (Mouse CD16/32)BD Biosciences553142
Fluorescence microscopeNikon ECLIPSE TE300
gentleMACS C Tubes (4 x 25 tubes)Miltenyi Biotec130-096-334
gentleMACS Octo Dissociator with HeatersMiltenyi Biotec130-096-427
Hanks' Balanced Salt Solution (HBSS) +CaCl2 +MgCl2 10xThermo Scientific14065049
Hanks' Balanced Salt Solution (HBSS) -CaCl2 -MgCl2 10xThermo Scientific14185045
iQ SYBR Green SupermixBio-rad1725006CUST
Iscript c-DNA synthesisBio-rad1708890
Latex beads, amine-modified polystyrene, fluorescent redSigma-AldrichL2776-1mL
Lipopolysaccharides (LPS) from Escherichia coli O55:B5Sigma-AldrichL2880
Macrophage-SFM serum-free mediumThermo Scientific12065074
MACS BSA Stock SolutionMiltenyi Biotec130-091-376
MACS SmartStrainers (70 μm), 4 x 25 pcsMiltenyi Biotec130-110-916
Mouse IgG1 PEMilliporeMABC002H
Mouse IgG2a PE Cy7Sony Biotechnology2601265
Mouse IL1 betaMiltenyi Biotec130-101-684
Multi-24 Column BlocksMiltenyi Biotec130-095-691
MultiMACS Cell24 SeparatorMiltenyi Biotec
Neural Tissue Dissociation Kit - PapainMiltenyi Biotec130-092-628
Nucleocounter NC-200Chemometec
Nucleospin RNA Plus XSMacherey Nagel740990.5
Nun EZFlip Top Conical Centrifuge TubesThermo Scientific362694
OPTILUX Petri dish - 100 x 20 mmDutscher353003
Pénicilline-streptomycine (10 000 U/mL)Thermo Scientific15140122
Rat IgG2b, k BV421BD Biosciences562603
Rat IgG2b, k BV510Sony Biotechnology2603230
REA control (S) PE vio 615Miltenyi Biotec130-104-616
REA control (S) Vio Bright B515Miltenyi Biotec130-113-445
Recombinant Mouse IFN-gamma ProteinR&D System485-MI
Recombinant Mouse IL-10 ProteinR&D System417-ML
Recombinant Mouse IL-4 ProteinR&D System404-ML
RIPA BufferSigma-AldrichR0278
Viability probe (FVS780)BD Biosciences565388

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