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 &#181;L DNase I (see table of materials) at 100 mg/mL (stock solution), final concentration 0.5 mg/mL.</li>
</ol>
2. Perfusion and Dissection
<ol>
	<li>Euthanize the mice with a lethal dose of pentobarbital (100 &#181;L at 400 mg/mL).</li>
	<li>Place the animal in supine position and wet the body with 70% ethanol.</li>
	<li>Cut the ventral skin with fine scissors just below the xyphoid process to expose the thoracic cavity.</li>
	<li>Make an incision in the diaphragm and cut the lateral aspects of the rib cage with fine scissorsin a caudal to rostral direction to expose the heart (and avoid cutting other organs). Identify the left ventricle (LV) and the right atrium (RA).</li>
	<li>Insert a butterfly catheter with a 25 G needle inside the LV. Make an incision on the RA with fine scissors and at the beginning of blood flow, start perfusion at 10 mL/min flow rate through the LV with 20 mL PBS to remove cells and blood from vessels; successful perfusion is noted by the blanching of the lungs and the liver as blood is displaced.</li>
	<li>Decapitate the animal with medium scissors. Dissect the spinal column with fine scissors and separate it from the body by excising the abdominal wall musculature on the ventral side of the spinal column and by cutting the vertebral column at the base of the tail.</li>
	<li>Insert a 10 mL syringe containing PBS and a mounted 200 &#181;L pipette tip into the lumbar side of the spinal column and flush the spinal cord on the cervical side. 
	NOTE: The 200 &#181;L pipette tip is cut with scissors at its widest end (approximately 1 cm) and tightly inserted on a 10 mL syringe previously filled with PBS.</li>
	<li>Remove the skin above the skull and use forceps to expose the brain. Insert a scissor blade at the base of the skull and cut the cranium following the sagittal suture. Insert small forceps into the cut and gently lift the skull. Dissect the brain from the mouse head and remove the olfactory bulb and cerebellum.</li>
	<li>Collect the brain and spinal cord in a 1.5 mL tube containing 1.128 mL PBS with digestion cocktail (from step 1.1).</li>
</ol>
3. Cell Dissociation
<ol>
	<li>Finely cut the brain and spinal cord in the 1.5 mL tube (step 2.9) into 1-2 mm3 pieces with small scissors (Figure 1).</li>
	<li>Incubate the 1.5 mL tube containing the minced tissues for 30 min in an incubator at 37 &#176;C with 5% CO2. 
	NOTE: Prepare the density gradient at this step.</li>
	<li>Add 20 &#181;L 0.5 M EDTA (stock solution), final concentration 10 mM, to the 1.5 mL tube to stop the enzymatic reaction.</li>
	<li>Gently make a cell suspension by pipetting up and down with a 5 mL pipette.</li>
	<li>Pass the cell suspension through a nylon mesh (70 &#181;m pore size) in a 50 mL tube using the plunger of a 10 mL syringe.</li>
	<li>Wash the nylon mesh with 20 mL 5% Fetal Bovine Serum (FBS)-PBS.</li>
	<li>Centrifuge for 7 min (300 x g) at room temperature (18 &#176;C). Discard the supernatant by aspiration and proceed to the next step. Be extremely careful when removing the supernatant; do not disturb the pellet, which can easily be aspirated by the aspirating pipette.</li>
</ol>
4. Density Gradient
<ol>
	<li>Prepare a stock solution of the density gradient medium by adding the appropriate amount of 10x PBS to the density gradient medium. 
	NOTE: Add one part 10X PBS to nine parts density gradient medium.</li>
	<li>Dilute the stock density gradient medium with 1x PBS for the different percentages as described in Table 1.</li>
	<li>Resuspend the pellet (from step 3.7) with 4 mL 30% density gradient medium and transfer to a 15 mL tube.</li>
	<li>Place a Pasteur pipette in the bottom of the 15 mL tube and slowly underlay 4 mL of 37% density gradient medium.</li>
	<li>Add 4 mL of 70% density gradient medium, as described above.</li>
	<li>Centrifuge the gradient for 40 min (800 x g) at room temperature (18 &#176;C). Ensure that the centrifuge stops with minimal or no brake so that the interphase is not disturbed. 
	NOTE: The tube will appear stratified. The cells layered at the 70% - 37% density gradient interface are the mononucleated immune cells (microglia and macrophage subpopulations); the upper levels are cell debris and myelin, respectively (Figure 2).</li>
	<li>Using a transfer pipette, gently remove the layer of myelin.</li>
	<li>Collect 3-4 mL of the 70-37% density gradient interphase containing the macrophage subpopulations cells into a clean 15 mL tube.</li>
	<li>Wash the cells 3x with PBS, total volume of 15 mL. Make sure that the density gradient medium containing the cells is diluted at least three times with PBS, and to centrifuge for 7 min (500 x g, 4 &#176;C) with the brake &#34;on&#34;.</li>
	<li>Resuspend the cell pellet with 1 mL PBS for cell labeling or for other functional assays.</li>
</ol>
5. Cell Labeling for Flow Cytometry
<ol>
	<li>Transfer the cells (step 4.10) into a flow cytometry tube.</li>
	<li>Add 1 &#181;L of fixable dead cell stain reagent to each cell sample obtained in step 4.10. Incubate for 30 min on ice, protected from light.</li>
	<li>Wash the cells once with 2 mL PBS and centrifuge for 5 min (300 x g, 4 &#176;C).</li>
	<li>Remove the supernatant, and resuspend the cell pellet with 400 &#181;L PBS 1% BSA, 0.1% azide.</li>
	<li>Add 1 &#181;g CD16/CD32 antibodies to 100 &#181;L of the cells to block the Fc receptors. Incubate 10-15 min on ice, protected from light.</li>
	<li>Prepare the primary antibody mix in PBS 1% BSA, 0.1% azide or PBS 1% BSA, 0.1% azide, 0.25% saponin, for cell permeabilization for intracellular staining.</li>
	<li>Add 100 &#181;L of primary antibodies mix (2x) and incubate for 30 min on ice, protected from light. 
	NOTE: All antibodies should be titrated prior to conducting the experiment to ensure optimal results.</li>
	<li>Wash the cells with 2 mL PBS and centrifuge for 5 min (300 x g, 4 &#176;C). Repeat this step three times.</li>
	<li>Add 100 &#181;L of a secondary antibody if the primary antibodies are not directly conjugated to a fluorophore, and incubate for 30 min on ice, protected from light.</li>
	<li>Wash the cells with 2 mL PBS and centrifuge for 5 min at 300 x g at 4 &#176;C). Repeat this step three times.</li>
	<li>Fix the cells with 100 &#181;L 1% PFA for 15 min at room temperature, protected from light. 
	NOTE: Caution: PFA is toxic. Use in a fume hood and wear personal protective equipment.</li>
	<li>Wash the cells with 2 mL PBS and centrifuge for 5 min at 300 x g at 4 &#176;C. Repeat this step three times.</li>
	<li>Resuspend the cell pellet with 200 &#181;L PBS and proceed to flow cytometry acquisition and analysis. Follow the gating strategy in Figure 3.</li>
</ol>

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The authors have nothing to disclose

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><tbody><tr><td>5 month-old Mice</td><td>Janvier</td><td>C57BL/6J</td><td></td></tr><tr><td>Liberase TL Research Grade</td><td>Sigma-Aldrich</td><td>5401020001</td><td>Digestion enzyme</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare Life Sciences</td><td>17-0891-01</td><td>Density gradient medium</td></tr><tr><td>Cell Strainer size 70 &amp;micro;m Nylon</td><td>Corning</td><td>731751</td><td></td></tr><tr><td>Venofix 25 G</td><td>BRAUN</td><td>4056370</td><td></td></tr><tr><td>Piston syringe 10 mL</td><td>Terumo</td><td>SS+10ES1</td><td></td></tr><tr><td>Pasteur pipette 230 mm</td><td>Dustcher</td><td>20420</td><td></td></tr><tr><td>1.5 mL tube</td><td>Eppendorf</td><td>0030 123.328</td><td></td></tr><tr><td>15 mL tube</td><td>TPP</td><td>91015</td><td></td></tr><tr><td>50 mL tube</td><td>TPP</td><td>91050</td><td></td></tr><tr><td>5 mL polystyrene round bottom tube</td><td>BD Falcon</td><td>352054</td><td></td></tr><tr><td>D-PBS (1x) without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>D-PBS (10x) without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Thermo Fisher Scientific</td><td>14200-067</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E4884</td><td></td></tr><tr><td>Bovine Serum Albumin solution 30%</td><td>Sigma-Aldrich</td><td>A7284</td><td></td></tr><tr><td>Paraformaldehyde 32% Solution</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>Caution -Toxic</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>PerCPCy5.5 Rat anti-mouse CD11b (clone M1/70)</td><td>eBioscience</td><td>45-0112</td><td></td></tr><tr><td>Rat IgG2b K Isotype Control PerCP-Cyanine5.5</td><td>eBioscience</td><td>45-4031</td><td></td></tr><tr><td>BV421 Rat anti-mouse CD45 (clone 30-F11)</td><td>BD Biosciences</td><td>563890</td><td></td></tr><tr><td>BV421 Rat IgG2b, &amp;kappa; Isotype Control RUO</td><td>BD Biosciences</td><td>562603</td><td></td></tr><tr><td>Rabbit anti-mouse TMEM119 (clone28 - 3)</td><td>Abcam</td><td>ab209064</td><td></td></tr><tr><td>AlexaFluor 647 Donkey anti-rabbit IgG</td><td>Life Technologies</td><td>A31573</td><td></td></tr><tr><td>Anti-Mouse CD16/CD32 Purified</td><td>eBioscience</td><td>14-0161</td><td>Mouse Fc Block</td></tr><tr><td>Fixable Dead Cell Stain Kits</td><td>Invitrogen</td><td>L34969</td><td></td></tr><tr><td>Mouse CCR2 APC-conjugated Antibody</td><td>R&amp;amp;D</td><td>FAB5538A</td><td></td></tr><tr><td>Rat IgG2B APC-conjugated Isotype Control</td><td>R&amp;amp;D</td><td>IC013A</td><td></td></tr><tr><td>Mouse CX3CR1 PE-conjugated Antibody</td><td>R&amp;amp;D</td><td>FAB5825P</td><td></td></tr><tr><td>Goat IgG PE-conjugated Antibody</td><td>R&amp;amp;D</td><td>IC108P</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804R</td><td></td></tr><tr><td>Cell analyzer</td><td>BD Biosciences</td><td>BD FACSVERSE</td><td></td></tr><tr><td>Data Analysis Software</td><td>FlowJo LLC</td><td>FlowJo</td><td></td></tr><tr><td>Fine scissors</td><td>F.S.T</td><td>14090-11</td><td></td></tr><tr><td>Standard Pattern Forceps</td><td>F.S.T</td><td>11000-13</td><td></td></tr><tr><td>Mayo Scissors</td><td>F.S.T</td><td>14010-15</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>F.S.T</td><td>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 ...

analysis-microglia-monocyte-derived-macrophages-from-central-nervous

Research

JoVE Journal

Immunology and Infection

23.8K Views.  Inserm U 1227, CNRS UMR 7225.  Dit protocol geeft een analyse van de macrofage subpopulaties in het centraal zenuwstelsel van het volwassen muis door middel van flowcytometrie en is nuttig voor het bestuderen van meerdere markers die door deze cellen worden uitgedrukt. 

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

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Neuroscience

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

Mouse Brain Histone Modification Quantification Using Flow Cytometry

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Author Spotlight: Enhancements in Gene Expression Regulation Research

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

Flow Cytometry-Based Assays for Microglial Engulfment of Synaptic Materials

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Author Spotlight: Advancing Understanding Through Technological Innovations in Psychoneuroimmunology

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

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

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