Name: analysis of microglia and monocyte-derived macrophages from the central nervous system by flow cytometry
Uploaded: 2017-06-22T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry at JoVE.com

This work was supported by grants from Agence Nationale pour la Recherche (ANR-12-MALZ-0003-02-P2X7RAD), Association France Alzheimer and Bpifrance. Our laboratory is also supported by Inserm, CNRS, Universit&#233; Pierre et Marie-Curie and the program &#34;Investissements d&#39;avenir&#34; ANR-10-IAIHU-06 (IHU-A-ICM).&#160;We would like to thank the assistance of the CELIS cell culture core facility.

All methods described here have been approved by the Institutional Animal Care and Use Committee at the ICM Institute and by the Darwin French Ethic Animal Committee, and are covered under the protocol 01407.02.
1. Preparation
<ol>
	<li>Prepare the digestion cocktail in a 1.5 mL tube by combining the following for each mouse: 1 mL Phosphate Buffered Saline (PBS); 123 &#181;L digestion enzyme (see table of materials) at 13 wunsch/mL (stock solution), final concentration 1.6 wunsch/mL; and 5 &...

<ol>
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It has been demonstrated that the microglia and MDM have different functions and phenotypes in the CNS, and thus the identification and the analysis of these macrophage subpopulations are essential in order to better understand neurological diseases9,18,25. Flow cytometry analysis using two markers (CD11b and CD45) allows for the distinction between each subpopulation (Figure 3C). This strategy was ...

The microglia are the parenchymal tissue-resident macrophages of the central nervous system (CNS). They play two key functional roles: immune defense and maintenance of the CNS homeostasis. In contrast to the MDM, which are renewed continually from the hematopoietic stem cells in the bone marrow, the microglial cells differentiate from primitive hematopoietic progenitor cells originating in the yolk sac (YS) that colonized the brain during embryonic development1,2,3. In rodents, the transcription factor Myb plays a crucial role in the development of all bone marrow derived monocytes and macrophages, but for YS derived microglia, this factor is dispensable and differentiation remains dependent on the transcription factor PU.14.
In the healthy CNS, microglia are dynamic cells that constantly sample their environment, scanning and surveying for invading pathogens or tissue damage5. The detection of such signals initiates a pathway to resolve the injury. The microglia rapidly switch from a ramified morphology to an amoeboid one, which is followed by phagocytosis and release of various mediators, such as pro- or anti-inflammatory cytokines. Thus, depending on their microenvironment, activated microglia can acquire a spectrum of distinct priming states6.
The microglia profoundly impact the development and progression of many neurological disorders. In the rodent models of Alzheimer&#39;s disease (AD)7, Amyotrophic Lateral Sclerosis (ALS)8, Multiple Sclerosis (MS)9 or Parkinson disease (PD)10, the microglia are shown to play a dual role, either inducing detrimental neurotoxicity or acting in a neuroprotective manner, which is dependent on the specific disease, the disease stage, and whether the disease was influenced by the systemic immune compartment7,8,9,10,11. Most of the CNS lesions observed in the diseases cited above contain a heterogeneous population of myeloid cells, including not only parenchymal microglia, but also perivascular and meningeal macrophages, as well as CNS-infiltrating MDM. These cell types may differentially contribute to the pathophysiologic mechanisms related to injury and repair7,12,13,14,15. The current challenge for investigators who are studying these disease models is to establish whether the peripheral monocytes and macrophages infiltrate the CNS and if so, to distinguish the resident microglia from these cells. Indeed, the microglial cells are very plastic; when they are activated, the microglia re-express markers that are usually expressed by peripheral monocytes and macrophages. The issue, therefore, relies on identifying markers that can distinguish the resident microglia from the infiltrating monocytes and macrophages.
The discrimination of these populations on brain slices by immunohistological applications is limited due to the lack of specific antibodies. However, flow cytometry analysis is an efficient technique to assess the expression of several markers and to distinguish cell populations (for example, lymphocytes, macrophages/MDM CD11b+CD45high, and microglia CD11b+CD45med), as well as cell subpopulations16,17,18. This protocol describes the procedures for isolating the mononuclear cells from the mouse CNS in neurologic disease models by using an optimized, enzymatic tissue dissociation and a density gradient centrifugation; as well as, a method for differentiating the microglia and MDM populations in the CNS by using flow cytometry.
Another approach is to eliminate the myelin and purify the cells by using magnetic beads conjugated to specific antibodies19,20,21. Myelin removal using anti-myelin magnetic beads is more expensive and affects the viability and the yield of isolated cells22. This step and the following immunomagnetic separation of the microglia, limit further studies of specific immune cell populations21,22.
These procedures provide an easy way to study the macrophage subpopulations in disease development, and to determine the drug effects or gene modifications on macrophage phenotypes and activation states.

<table border="0" cellpadding="0" cellspacing="0" width="908">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">5-month-old Mice</td>
			<td style="width:223px;">Janvier</td>
			<td style="width:119px;">C57BL/6J</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Liberase TL Research Grade</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">5401020001</td>
			<td>Digestion enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Deoxyribonuclease I from bovine pancreas</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">DN25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Percoll</td>
			<td style="width:223px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0891-01&#160;</td>
			<td>Density gradient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Cell Strainer size 70 &#181;m Nylon</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">731751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Venofix 25G</td>
			<td style="width:223px;">BRAUN</td>
			<td style="width:119px;">4056370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Piston syringe 10 mL</td>
			<td style="width:223px;">Terumo</td>
			<td style="width:119px;">SS+10ES1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Pasteur pipette 230 mm</td>
			<td style="width:223px;">Dustcher</td>
			<td style="width:119px;">20420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">1,5 mL&#160; tube</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:119px;">0030 123.328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">15 mL&#160; tube</td>
			<td style="width:223px;">TPP</td>
			<td style="width:119px;">91015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">50 mL&#160; tube</td>
			<td style="width:223px;">TPP</td>
			<td style="width:119px;">91050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">5 mL polystyrene round bottom tube</td>
			<td style="width:223px;">BD Falcon</td>
			<td style="width:119px;">352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">D-PBS (1X) without Ca2+/Mg2+</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">D-PBS (10X) without Ca2+/Mg2+</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14200-067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fetal bovine serum</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Bovine Serum Albumin solution 30%</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Paraformaldehyde 32% Solution</td>
			<td style="width:223px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714-S</td>
			<td>Caution -Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Saponin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Sodium Azide</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">47036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">PerCPCy5.5 Rat anti-mouse CD11b (clone M1/70)</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">45-0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rat IgG2b K Isotype Control PerCP-Cyanine5.5</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">45-4031&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BV421 Rat anti-mouse CD45 (clone 30-F11)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">563890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BV421 Rat IgG2b, &#954; Isotype Control RUO</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">562603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rabbit anti-mouse TMEM119 (clone28-3)</td>
			<td style="width:223px;">Abcam</td>
			<td style="width:119px;">ab209064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">AlexaFluor 647 Donkey anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A31573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Anti-Mouse CD16/CD32 Purified</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">14-0161</td>
			<td>Mouse Fc Block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fixable Dead Cell Stain Kits</td>
			<td style="width:223px;">Invitrogen</td>
			<td style="width:119px;">L34969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mouse CCR2 APC-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">FAB5538A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rat IgG2B APC-conjugated Isotype Control</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">IC013A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mouse CX3CR1 PE-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">FAB5825P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Goat IgG PE-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">IC108P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Centrifuge</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:119px;">5804R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Cell analyzer</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">BD FACSVERSE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Data Analysis Software</td>
			<td style="width:223px;">FlowJo LLC</td>
			<td style="width:119px;">FlowJo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fine scissors</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">14090-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Standard Pattern Forceps</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">11000-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mayo Scissors</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">14010-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Dumont #5 Forceps</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of microglia and monocyte-derived macrophages from the central nervous system by flow cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

After the density gradient centrifugation and antibody staining, the cells were acquired on a flow cytometer and analyzed using a morphological gating strategy as follows. A first gate was defined in the dot plot Forward-Scattered-Area (FSC-A) versus Forward-Scattered-Height (FSC-H) to discriminate single cells from doublets (Figure 3A). The single cells were then gated on FSC-A versusSide-Scattered-Area (SSC-A) dot plots to exclude cell deb...

Watch this Scientific Journal Video about Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry at JoVE.com

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

The overall goal of this experiment is to assess the various markers expressed by macrophage subpopulations within the central nervous system or CNS. This method can help answer key questions in the neuroimmunology field, such as duperry macrophages as we try to CNS doing dizzies, and what are the phenotypes of CNS in macrophages. The main advantages of this technique are the isolation of a large number of highly purified macrophage sub-population, as well as a contification of macrophage sub-population cell marker.Begin by using fine scissors to cut the ventral skin just below the xiphoid process to expose the thoracic cavity. Make an incision in the diaphragm, and cut the lateral aspects of the ribcage in a caudal to rostral direction to expose the heart. After identifying the left ventricle and the right atrium, insert a butterfly catheter with a 25 gauage needle into the left ventricle.Make an incision on the right atrium and begin profusing 20 milliliters of PBS through the catheter at a rate of ten milliliters per minute as soon as the blood begins to flow. A successful profusion will be indicated by a blanching of the lungs and the liver as the blood is displaced. Next, use fine scissors to dissect the spinal column.Excising the abdominal wall musculature on the ventral side of the spinal column, and cutting the vertebral column at the base of the tail to remove the spine as it is harvested. Insert a ten milliliter syringe of PBS equipped with a modified 200 microliter pipette tip into the lumbar side of the spinal column. Then flush the spinal cord on the cervical side.Remove the skin above the skull, and insert one tip of the scissors at the base of the skull. Cut the cranium following the sagittal suture. Then insert small forceps into the cut and gently remove the top of the skull.Dissect the brain from the mouse head and remove the olfactory bulb and cerebellum. Then place the brain and spinal cord in a one point five milliliter tube containing one point one two eight milliliters of digestion cocktail. To dissociate the cells, use small scissors to finely cut the brain and spinal cord into one to two cubic millimeter pieces and place the pieces at 37 degrees Celsius and five percent Carbon Dioxide for 30 minutes.At the end of the incubation, stop the digestion with 20 microliters of zero point five molar EDTA, followed by gentle trituration with a five milliliter pipette. Filter the resulting cell suspension through a 70 micron mesh strainer into a 50 milliliter conical tube using the plunger of a ten milliliter syringe to gently macerates any remaining brain or spinal cord tissue. Rinse the strainer with 20 milliliters of five percent FBS and PBS.And collect the filtered cells by centrifugation. Then, carefully aspirate the supernatant without disturbing the pellet. For density gradient separation of the cells, we suspend the pellet with four milliliters of 30 percent density gradient medium, and transferred the cells into a 15 milliliter conical tube.Gently underlay four milliliters of 37 percent density gradient medium, followed by four milliliters of 70 percent density gradient medium under the 37 percent density gradient medium. Transfer the tube to a bench-top centrifuge to separate the cells. The microglia and macrophage cells will be at the interface of the 70 and 37 percent density gradient layers.The tube will appear stratified. After carefully removing the upper layer of myelin, transfer three to four milliliters of the microglia macrophage interphase layer into a new 15 milliliter tube. Then wash the immune cells with three centrifuges in 15 milliliters of PBS, re-suspending the pellet in one milliliter of PBS after the final wash.To asses the macrophage sub-population marker expression, transfer the cells into a five milliliter flow cytometry analysis tube, followed by the addition of one microliter of fixable dead cell stain reagent. After 30 minutes on ice, protected from light, wash the samples in two milliliters of PBS, and re-suspend the pellet in 400 microliters of fax buffer per experimental analysis tube. Split the cells into the appropriate number of five milliliter flow cytometry analysis tubes.Then block the non-specific FC receptor binding with one microgram of CD 16 CD 32 blocking antibody cocktail per 100 microliters of cells for a 10 to 15 minute incubation on ice, protected from light. Next, label the cells with 100 microliters of the appropriate primary antibodies, for a 30 minute incubation on ice, protected from light. At the end of the incubation, wash the cells in two milliliters of PBS four times.Then add 100 microliters of the appropriate secondary antibodies for 30 minutes on ice, protected from light, followed by four washes in PBS as just demonstrated. After the last wash, gently re-suspend the pellets in 100 microliters of one percent paraformaldehyde for 15 minutes at room temperature, protected from light. Then wash the cells four times in PBS.Re-suspend the final pellets in 200 microliters of fresh PBS, and analyze the cells by flow cytometry. Brain-and spinal cord-derived microglia and monocyte-derived macrophages are identified based on their CD 11 B and CD 45 expression. The expression of CX3CR1, and Tmem119, by the CD 11 B positive CD 45 medium microglial cells allows this cell population to be further distinguished from the CD 11 C positives CD 45 high monocyte-derived macrophages, which do not express either of these markers, but do express CCR2.After density gradient separation, 85 to 95 percent of the cells are still viable. In the absence of density gradient isolation, the macrophage subpopulations are not identifiable in a forward bi-side scatter plot, because of the presence of non-macrophage cells, and myelin debris. As the epitope recognized by the anti-Tmem119 antibody is intracellular, permeabilization is required for labeling of this important microglial cell marker.Note that permeabilization induces cell shrinkage, however, causing the macrophage subpopulations to appear smaller by forward versus side scatter analysis. Once mastered, this technique can be completed in five hours if it is performed properly. While I attempted this procedure, it's important to remember to set up the density gradiance very carefully.Following this procedure, other methods, like quantitative rt-PCR can be performed to answer additional questions about macrophage subpopulation signaling pathways. After its development, this technique paved the way for researchers in neuroscience field to explore the activation of macrophage subpopulation in mouse model of neurologic disease. After watching this video, you should either conduct testing or follow this tradition as macrophage subpopulations by flow cytometry.Don't forget that working with paraformaldehyde can be extremely hazardous, and that precautions such as using a hood should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...