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Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia

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Abstract

Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

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Abbreviations

chemokine C-C motif ligand 1:

CCL1, aka SCYA1, I-309, TCA3, P500, SISe

chemokine C-C motif ligand 2:

CCL2, aka MCP-1, MCP1, GDCF-2, HC11, HSMCR30, MCAF, SCYA2, SMC-CF

chemokine C-C motif ligand 3:

CCL3, aka G0S19-1, LD78ALPHA, MIP-1α, MIP1A, SCYA3

chemokine C-C motif ligand 4:

CCL4, aka ACT2, AT744.1, G-26, HC21, LAG-1, LAG1, MIP-1β, MIP1B, MIP1B1, SCYA2, SCYA4

chemokine C-C motif ligand 5:

CCL5, aka D17S136E, RANTES, SCYA5, SIS-delta, SISd, TCP228, eoCP

complement 5a receptor:

C5AR1, aka CD88

cAMP:

cyclic AMP

cluster of differentiation 47:

CD47, aka IAP, MER6, OA3

cluster of differentiation 200:

CD200, aka MOX1, MOX2, MRC, OX-2

cluster of differentiation 200 receptor:

CD200R1, aka CD200R, HCRTR2, MOX2R, OX2R

CX3CL1:

Chemokine C-X3-C motif ligand, aka fractalkine, neurotactin, ABCD-3, C3Xkine, CXC3, CXC3C, NTN, NTT, SCYD1

CX3CR1:

CX3C chemokine receptor 1; aka fractalkine receptor, CCRL1, CMKBRL1, CMKDR1, GPR13, GPRV28, V28

Dock180, aka Dock1:

Dedicator of cytokinesis 180

Dok1, aka P62DOK:

Docking protein 1

Dok2, aka p56DOK, p56dok-2:

Docking protein 2

ERK1/2:

Extracellular signal-regulated protein kinases 1 and 2

Fcgr1:

High affinity immunoglobulin gamma Fc receptor I

Fcgr3:

Low affinity immunoglobulin gamma Fc region receptor III

ITAM:

Immunoreceptor tyrosine-based activation motif

ITIM:

Immunoreceptor tyrosine-based inhibitory motif

IP3 :

Inositol triphosphate

IL-1:

Interleukin 1

MMP3:

Matrix metalloproteinase-3

MFG-e8:

Milk fat globule-EGF factor 8 protein

PLC:

Phospholipase C

NFκB:

Nuclear factor kappa-light-chain-enhancer of activated B cells

PTPN6, aka HCP, HCPH, HPTP1C, PTP-1C, SH-PTP1, SHP-1, SHP-1 L, SHP1:

Protein tyrosine phosphatase, non-receptor type 6

PTPN11, aka BPTP3, CFC, NS1, PTP-1D, PTP2C, SH-PTP2, SH-PTP3, SHP2:

Protein tyrosine phosphatase, non-receptor type 11

PKA:

Protein kinase A

PTK:

Protein tyrosine kinase

P2RX4, aka P2X4, P2X4R:

Purinergic receptor P2X, ligand-gated ion channel, 4

P2RY6, aka P2Y6:

Pyrimidinergic receptor P2Y, G-protein coupled, 6

P2RY12, aka ADPG-R, BDPLT8, HORK3, P2T(AC), P2Y(12)R, P2Y(AC), P2Y(ADP), P2Y(cyc), P2Y12, SP1999:

Purinergic receptor P2Y, G-protein coupled, 12

Rac1:

Ras-related C3 botulinum toxin substrate 1

SIRPα, aka SIRP, SHPS1, CD172A, PTPNS1, BIT, MFR, MYD-1, P84:

Signal regulatory protein α

Syk:

Spleen tyrosine kinase

Trem2:

Triggering receptor expressed on myeloid cells 2

Tyrobp:

TYRO protein tyrosine kinase-binding protein

VASP:

Vasodilator-stimulated phosphoprotein

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Acknowledgments

This work was supported by the National Institutes of Health – National Institute on Aging (NIH-NIA) AG031158 (SJB), the Medical Student Training in Aging Research (MSTAR) program (TAV, CDR), and startup funds from the University of Nebraska Medical Center (SJB). We thank Tammy L. Kielian, Ph.D. for reviewing the draft manuscript; Megan L. Michalak, Victoria B. Smith, and Charles A. Kuszynski, Ph.D. of the UNMC cell analysis facility for their assistance performing the flow cytometry studies.

Conflict of Interest

The authors declare no conflicts of interest for this work.

Author Contributions

All studies conceived, analyzed, reported by SJB. TAV, CDR, TAH performed and analyzed flow cytometry and in vitro microglial studies. TAH, JA collected and analyzed DIC and confocal microscopy images demonstrating microglial enrichment. TAV, TAH, JA assisted in manuscript preparation.

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Authors and Affiliations

  1. Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198, USA

    T. A. Volden, C. D. Reyelts, T. A. Hoke & S. J. Bonasera

  2. Developmental Neuroscience, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, NE, 68198, USA

    J. Arikkath

  3. University of Nebraska Medical Center, 3028 Durham Research Center II, Omaha, NE, 68198-5039, USA

    S. J. Bonasera

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  1. T. A. Volden
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  2. C. D. Reyelts
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  3. T. A. Hoke
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  4. J. Arikkath
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  5. S. J. Bonasera
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Correspondence to S. J. Bonasera.

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Volden, T.A., Reyelts, C.D., Hoke, T.A. et al. Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia. J Neuroimmune Pharmacol 10, 655–665 (2015). https://doi.org/10.1007/s11481-015-9628-7

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