Isolation and Analysis of Mouse Microglial Cells

Jenny A. Garcia1, Sandra M. Cardona1, Astrid E. Cardona1

1 The University of Texas at San Antonio, San Antonio, Texas
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.35
DOI:  10.1002/0471142735.im1435s104
Online Posting Date:  February, 2014
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Abstract

Microglia are mononuclear phagocytes that make up about 10% of the central nervous system (CNS). They are known for their surveillant behavior, which involves continuous monitoring of neural tissue by extending and retracting their processes. Microglial cells are derived from myeloid progenitor cells and play important roles in homeostasis as well as inflammatory and immune responses in the brain. This unit describes several microglial cell isolation protocols that can be easily adapted for projects requiring a rapid and efficient analysis of mouse microglial cells by flow cytometry. Methods for visualizing microglial cells using in situ immunohistochemistry and immunochemistry in free‐floating sections are also included. Curr. Protoc. Immunol. 104:14.35.1‐14.35.15. © 2014 by John Wiley & Sons, Inc.

Keywords: mononuclear cells; microglial isolation; immunostaining; CX3CR1/GFP; CNS; myeloid cells; microglia; Percoll gradients; flow cytometry; histochemistry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Leukocytes Containing Microglial Cells from Adult Mouse CNS Tissues
  • Alternate Protocol 1: Enzymatic Neural Tissue Dissociation
  • Basic Protocol 2: CD11b Enrichment of Microglial Cells
  • Support Protocol 1: Cell Surface Staining and Flow Cytometric Analysis
  • Basic Protocol 3: Isolation of CNS Tissues for In Situ Immunocytochemistry
  • Basic Protocol 4: Immunocytochemistry in Free‐Floating Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Leukocytes Containing Microglial Cells from Adult Mouse CNS Tissues

  Materials
  • Donor mice: C57BL6, 7 to 9 weeks old, male or female (The Jackson Laboratory, cat. no. 00664)
  • Isoflurane
  • O 2 source
  • 1× HBSS (Ca2+‐ and Mg2+‐free; Life Technologies, cat. no.14175), ice cold
  • RPMI 1640 medium supplemented with L‐glutamine, without phenol red (Life Technologies, cat. No. 11835).
  • 100% and 70% Percoll (see reciperecipes)
  • 1× HBSS/10 mM HEPES (see recipe)
  • Cell staining buffer (Biolegend, cat. no. 420201)
  • Isoflurane induction chamber (e.g., Harvard Apparatus; also see unit 1.4)
  • Dissecting instruments
  • 6‐well tissue culture plates
  • 7‐ml Dounce homogenizers with A (loose) and B (tight) pestles (Pyrex, Wheaton)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Refrigerated centrifuge (Eppendorf, model no. 5810 R) or similar
  • Additional reagents and equipment for anesthesia of rodents (unit 2.NaN) and perfusion (unit 15.1)

Alternate Protocol 1: Enzymatic Neural Tissue Dissociation

  Additional Materials (also see protocol 1)
  • Neural Tissue Dissociation Kit (Papain) (Miltenyi Biotec, cat. no. 130‐092‐628)
  • 1× HBSS containing Ca2+ and Mg2+ (Life Technologies, cat. no. 14025134; alternately, PBS containing Ca2+ and Mg2+ can be used, Hyclone, cat. no. SH30264.01)
  • Myelin removal buffer (see recipe)
  • Razor blade
  • C tubes (Miltenyi Biotec, cat. No. 130‐093‐237)
  • gentleMACS Dissociator (Miltenyi Biotec, cat. No 130‐093‐235) or equivalent
  • 37°C incubator with rotating tube holders
  • 40‐µm nylon mesh cell strainers
  • 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Additional reagents and equipment for isolation of CNS tissues ( protocol 1, steps 1 and 2)

Basic Protocol 2: CD11b Enrichment of Microglial Cells

  Materials
  • Cell suspension ( protocol 1 or protocol 2Alternate Protocol)
  • Myelin Removal Beads II (Miltenyi Biotec, cat. No. 130‐096‐733)
  • Myelin removal buffer (see recipe)
  • CD11b (Microglia) MicroBeads (Miltenyi Biotec, cat. No. 130‐093‐634)
  • CD11b enrichment buffer (see recipe)
  • 5‐ml polystyrene tubes
  • LS columns (Miltenyi Biotec, cat. No. 130‐042‐401)
  • MidiMACS Separator (Miltenyi Biotec, cat. No. 130‐402‐302)
  • Refrigerated centrifuge
  • MS columns (Miltenyi Biotec, cat. No. 130‐402‐201; for total cell numbers up to 2 × 108)
  • OctoMACS Separator (Miltenyi Biotec, cat. No.130‐042‐109)
  • 15‐ml conical tubes
  • Additional reagents and equipment for counting viable cells using trypan blue exclusion (APPENDICES & )

Support Protocol 1: Cell Surface Staining and Flow Cytometric Analysis

  Materials
  • Mice to isolate blood and prepare single‐stain controls for flow cytometry (any wild‐type strain without endogenous expression of fluorescent proteins can be used, e.g. C57BL6 mice, The Jackson Laboratory, cat. no. 00664 or SJL/J, Harlan, cat. no. 052)
  • 10× HBSS (Ca2+ and Mg2+‐free; Life Technologies, cat. No. 14185)
  • 1× HBSS/10 mM HEPES (see recipe)
  • CD11b‐enriched cell samples (experimental sample obtained from protocol 3)
  • FC block: rat anti‐mouse CD16/CD32 clone 2.4G2 in cell staining buffer (BD Pharmingen)
  • Cell staining buffer (Biolegend, cat. no. 420201)
  • Antibodies (Table 14.35.1), fluorescently labeled (unit 5.3)
  • 4% paraformaldehyde (see recipe)
  • Sterile 5‐mm Goldenrod Animal Lancets
  • Refrigerated centrifuge
  • 15 ml conical tubes
  • LSR II flow cytometer (BD Biosciences) or equivalent (also see Chapter 5)
  • FlowJo analysis software (http://flowjo.com/)
  • Additional reagents and equipment for blood collection from mice (unit 1.7) and flow cytometry (Chapter 5)
Table 4.5.1   MaterialsCommon Antibodies Used for Analysis of Microglial Cells by Flow Cytometry

Antibody markers a Clone
CD45 30‐F11
CD11b (microglia) M1/70
CD11c (dendritic cells) N418
CD80 16‐10A1
CD86 GL‐1
I‐A/I‐E (MHC II) M5/114.15.2
CD274 (PD‐L1) 10F.9G2
F4/80 (macrophage/blood monocytes) BM8
Ly6C (macrophage) ER‐MP20
CD115 (monocyte, macrophage) AFS98
Ki‐67 (cell proliferation), intracellular B56

 aAntibodies can be purchased labeled with fluorochrome of choice; alternately unlabeled antibodies can be labeled as in unit 1.7. Combination of fluorochromes must be done based on the capabilities of your flow cytometry analyzer.

Basic Protocol 3: Isolation of CNS Tissues for In Situ Immunocytochemistry

  Materials
  • Mice, control and experimental (wild‐type, transgenic, or knockout depending on the particular experiment and model used, >3 weeks old)
  • Isoflurane
  • O 2 source
  • 1× HBSS (Life Technologies, cat. No. 14175)
  • 4% paraformaldehyde (see recipe)
  • Cryoprotection solution (see recipe)
  • Absolute ethanol
  • 30% (w/v) sucrose in PBS (see appendix 1044 for PBS)
  • Cryostorage solution (see recipe)
  • Isoflurane induction chamber (e.g., Harvard Apparatus; also see unit 1.4)
  • Dissecting instruments
  • Perfusion pump
  • Multi‐platform shaker
  • Freezing microtome (Leica, model SM 2010R) or similar
  • 22‐cm‐long Profile C Microtome blade (Leica Biosystems, cat. no. 14021607116)
  • 24‐well plates
  • Additional reagents and equipment for anesthesia of rodents (unit 15.1), perfusion (unit 21.4), and immunohistochemistry (unit 15.1)

Basic Protocol 4: Immunocytochemistry in Free‐Floating Tissue Sections

  Materials
  • 30‐ to 50‐µm sections from well perfused mice ( protocol 5)
  • 1× Dulbecco's phosphate‐buffered saline (DPBS), modified (HyClone, cat. no. SH30264.01)
  • Antigen retrieval solution (Dako, cat. No. S1699; optional)
  • Blocking solution (see recipe)
  • DPBS (see above) containing 0.1% (v/v) Triton X‐100
  • Primary antibody: rabbit anti‐Iba‐1 (Wako, cat. no. 01919741), diluted 1:4000 in DPBS/0.1% Triton X‐100
  • Secondary antibody: biotin‐conjugated goat anti‐rabbit (Vector Labs, cat. no. BA‐1000) diluted 1:1000 in DPBS/0.1% Triton X‐100
  • Avidin/biotin complex (ABC; Vector Labs, cat. no. PK‐6100), diluted 1:400 in DPBS/0.1% Triton X‐100
  • DAB (Invitrogen, cat. no. 750118); aliquot and store at −20°C
  • Xylenes
  • Permount (Fisher Scientific) or Prolong (Life Technologies) mounting medium
  • Paint brush
  • Glass reservoir
  • 9‐well clear glass spot plate
  • Multi‐platform shaker
  • Stereomicroscope
  • Superfrost microscope slides
  • 65°C oven
  • Coverslips
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Figures

Videos

Literature Cited

Literature Cited
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  Ford, A.L., Goodsall, A.L., Hickey, W.F., and Sedgwick, J.D. 1995. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein‐reactive CD4+ T cells compared. J Immunol. 154:4309‐4321.
  Jung, S., Aliberti, J., Graemmel, P., Sunshine, M.J., Kreutzberg, G.W., Sher, A., and Littman, D.R. 2000. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol. Cell Biol. 20:4106‐4114.
  Landsman, L., Bar‐On, L., Zernecke, A., Kim, K.W., Krauthgamer, R., Shagdarsuren, E., Lira, S.A., Weissman, I.L., Weber, C., and Jung, S. 2009. CX3CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival. Blood 113:963‐972.
  Mizutani, M., Pino, P.A., Saederup, N., Charo, I.F., Ransohoff, R.M., and Cardona, A.E. 2012. The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood. J. Immunol. 188: 29‐36.
  Ponomarev, E.D., Shriver, L.P., Maresz, K., and Dittel, B.N. 2005. Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J. Neurosci. Res. 81:374‐389.
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