This work was funded by the US Department of Veterans Affairs BLR&amp;D IK2 BX001686 (to TAB), and grants from the University of Minnesota Healthy Foods, Healthy Lives Institute (to CMD, JPN, and TAB) and the Minnesota Veterans Medical Research &amp; Education Foundation (to TAB). We thank Drs. Philippe Marambaud (Feinstein Institute for Medical Research, Manhasset, NY) and Weihua Zhao (Methodist Hospital, Houston, TX) for providing the BV2 cell line.

1.小胶质细胞培养维护<ol><li>暖细胞培养基（Dulbecco氏改良的Eagle培养基; DMEM）中补充有10％胎牛血清（FBS）和1％青霉素/链霉素/新霉素（PSN）至37℃。 </li><li>获取BV2小胶质细胞的冷冻库存的通道18 - 在-80℃，25从存储中。迅速解冻在37℃水浴细胞。 </li><li>轻轻细胞转移到含有10ml细胞培养基一个75cm 2的排出烧瓶。 </li><li>在37℃孵育烧瓶在5％CO 2。吸媒体后24小时，并用新鲜的培养基替换。培养细胞，直到大约70 - 80％汇合。 </li></ol> 2.计数和电镀细胞<ol><li>在37℃水浴，温DMEM和1x-胰蛋白酶EDTA溶液。 </li><li>从细胞吸媒体和加入500μl胰蛋白酶。孵育在37℃，2 - 5分钟。 </li><li>使用刮刀从烧瓶除去细胞并在5ml悬的DMEM。通过一个70微米的过滤器的细胞放入50毫升管三次。 </li><li>算使用血球细胞和种子8000个细胞/孔在黑色在补充有10％FBS和1％的PSN200μl的DMEM中，终体积壁透明底板。在37℃下在5％CO 2下孵育板24小时。 </li><li>移除旧的DMEM和用200μl温热的DMEM补充有1％的PSN血清饥饿的小胶质细胞替换。在37℃下在5％CO 2下孵育板24小时。 </li></ol> 3.激活小胶质细胞<ol><li>在单独的管中，稀AGNPS（0.01，0.05或0.1微克/毫升）和车辆/空档控制（柠檬酸钠，0.04毫摩尔）在补充有1％的PSN至最终工作浓度无血清DMEM。 </li><li>从每孔中取出100微升媒体和100微升适当的治疗化合物的替代。 </li><li>在37℃下在5％CO 2下孵育板24小时。 </li></ol> 4.网络滤波条件培养基<ol><li>收集200μl的培养基上清液从每孔中，并转移到与收集管（1.5至2ml容量）的过滤器（截留分子量为10kDa的）。 </li><li>以14,000 xg离心离心在室温下15分钟。 </li><li>丢弃流过（上清液与颗粒），然后将过滤器倒置到一个新的收集管。 </li><li>在1000 xg离心离心在室温下进行2分钟。 注:将所得的过滤介质（含有分泌的细胞因子）由六倍浓缩。 </li><li>使浓缩物至400微升体积用补充有10％FBS和1％的PSN和存储在冰上新鲜DMEM中。 注:为了确保剩余AGNPS从过滤介质去除，生成基于该特征峰的AgNP浓度在约390 - 420纳米10。简单地说，（如在步骤3.1中描述0，0.01，0.025，0.05,0.1和0.2微克/毫升），并过滤制备未过滤AGNPS等份车辆控制（柠檬酸钠，0.04毫摩尔）在无血清的DMEM中。等份加入50μl过滤样品和标准的成黑壁透明底板。在390使用分光光度计，测定吸光度 - 410 nm和比较读数到AgNP浓度曲线。如果滤波的样本具有相同的吸光度的值作为车辆控制样品，则可以假定残余AGNPS被除去。 </li><li>使用一半的过滤介质，从市售的试剂盒测定TNF-α的分泌以下说明。 </li></ol> 5.下丘脑细胞培养维护<ol><li>温暖的DMEM细胞培养基中补充有10％FBS和1％的PSN至37℃。 </li><li>获得下丘脑的冷冻股票（mHypo-A1 / 2）通过18细胞 - 25保存在80℃。迅速解冻在37℃水浴细胞。 </li><li>轻轻细胞转移到含有10ml细胞培养基一个75cm 2的排出烧瓶。 </li><li>在5％孵育烧瓶CO 2的</sUB&gt; 37℃。吸媒体后24小时，并用新鲜的培养基替换。培养细胞，直到大约在70％ - 80％汇合。 </li></ol> 6.确定下丘脑细胞毒性<ol><li>温暖的DMEM，并在37℃水浴中1×胰蛋白酶-EDTA溶液。 </li><li>从下丘脑细胞吸媒体和加入500μl胰蛋白酶。孵育在37℃，2 - 5分钟。除去使用如步骤2如上所述刮刀从烧瓶的细胞。 </li><li>算使用血球细胞中并在5000个细胞/孔在黑色在补充有10％FBS和1％的PSN200μl的DMEM中，终体积壁透明底板板丘脑细胞中并在37℃在5％CO 2孵育过夜。 </li><li>取出100微升使用多道移液器旧媒体，并添加100微升过滤条件集中媒体，为1X终浓度。 </li><li>在37℃下在5％CO 2下孵育板24小时。 </li><li>添加221;刃天青试剂l的，并在37℃下孵育板在5％CO 2 20分钟。 </li><li>使用多模分光光度计记录荧光（560纳米EX / 590nm处的EM）来测量细胞活力。报告结果为相对荧光单位（RFU）。 </li></ol>

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The authors have no conflicts to disclose.

Recent studies support that environmental exposure contributes to obesity and other neurodegenerative diseases 11,12. However, techniques used in previous studies are time consuming and expensive. Economic considerations, physiologically relevant delivery systems, ethical issues with extensive use of in vivo animal models, and difficulty translating findings into meaningful health advisories are a few of the major challenges that have impeded advancements in studying NP-induced neurotoxicity 13</...

环境污染，特别是那些纳米粒子（NP）范围（1 - 20纳米的直径），已与肥胖症和由于跨越血脑屏障1-3的能力其它神经变性疾病。升高的接触污染可能诱发炎症在中枢神经系统，包括下丘脑1。在发生这种情况可能是通过小胶质细胞的纳米颗粒诱导的激活（脑免疫细胞）4-一个潜在机制。以前的研究已经在体内模型中用于研究纳米颗粒对脑健康这是费时的，昂贵的影响，并且不直接回答的纳米颗粒如何影响小胶质细胞的问题。小胶质细胞在中枢神经系统中发挥着多方面的作用，包括维护脑微环境，并通过的分泌因子和细胞因子的释放周围的神经元进行通信。根据不同的刺激，小胶质细胞可激活到M1的公关邻炎症或M2抗炎状态。例如，M1激活的小胶质细胞释放促炎细胞因子如肿瘤坏死因子α（TNF-α），而M 2活化的小胶质细胞释放抗炎细胞因子，包括白细胞介素-4（IL-4）。为了验证我们的体外生物传感器替代确定空气污染物的毒性，我们测量到20纳米的银纳米颗粒（AGNPS）小胶质细胞的反应。本文的目的是描述一种体外小胶质细胞系如何可以用作用于测试小鼠小神经胶质响应于NP和如何小胶质细胞活化的影响下丘脑的细胞的替代生物传感器标记。长期打算在验证模型的应用是在测试大脑健康和神经退行性疾病现实世界的污染物的影响。我们提供一种在体外 96孔格式测定的详细说明，用于测量小胶质细胞活化和下丘脑细胞存活麦克风的曝光以下<html> roglial条件培养基。 测定小胶质细胞活化使用酶联免疫吸附测定（ELISA）以下的TNF-α的酶下列AgNP曝光。以确定对下丘脑细胞中激活的小胶质细胞的效果，AGNPS被使用的过滤装置的小胶质细胞上清液（条件培养基）中除去。该过滤装置保持细胞因子，同时排除根据大小AGNPS。简要地说，从具有或不AGNPS治疗小神经胶质细胞收集上清液，加至过滤器，并在14000×g离心15分钟。那时我们能够确定对下丘脑细胞活力小胶质细胞分泌的细胞因子的影响。如前所述5,6-暴露于条件培养基（含有细胞因子）的细胞毒性通过基于刃天青的测定法来确定。代谢活性细胞减少刃天青并产生正比于活细胞7的数量的荧光信号。 核苷酸"&gt;有使用这种技术比其他（如共培养，反式好设置，或在体内实验）的多个优点。我们的模型提供了直接激活的小胶质细胞，并确定是否分泌的因子是有毒的神经元8的能力。目前的协议采用永生BV2小胶质细胞刺激与20纳米的直径的纳米颗粒，和永生化的鼠下丘脑细胞（指定mHypo-A1 / 2）9，用于测定随后的反应。虽然该协议已经为这些特定的条件进行了优化，该方法可以是改变，以在小胶质诱导的细胞死亡的其它模型一起使用，或与其它类型的细胞包括原代小神经胶质细胞和神经元。

<table><tbody><tr><td>&lt;strong&gt;Cells/Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse microglial cell line (BV2)</td><td>Interlab Cell Line Collection (Genoa, Italy)</td><td>ATL03001</td><td></td></tr><tr><td>Adult Mouse Hypothalamus Cell Line mHypoA-1/2&amp;nbsp;</td><td>Cellutions Biosystems Inc.</td><td>CLU172</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium</td><td>Invitrogen</td><td>10313-039</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>PAA Labs</td><td>A15-751</td><td></td></tr><tr><td>Penicillin/Streptomycin/Neomycin</td><td>Thermo Fisher Scientific</td><td>15640-055</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Silver nanoparticles (20 nm)</td><td>Sigma-Aldrich</td><td>730793</td><td></td></tr><tr><td>&lt;br/&gt; PrestoBlue Cell Viability Reagent</td><td>Invitrogen</td><td>A13262</td><td></td></tr><tr><td>Mouse TNF-&amp;alpha; ELISA Max Delux</td><td>Biolegend</td><td>430904</td><td></td></tr><tr><td>Lipopolysaccharide</td><td>Sigma-Aldrich</td><td>L4391</td><td></td></tr><tr><td>Sodium Citrate</td><td>Sigma-Aldrich</td><td>S4641</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96 W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile</td><td>Thermo Fisher Scientific</td><td>165305</td><td></td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane</td><td>EMD Millipore</td><td>UFC501008</td><td></td></tr><tr><td>SpectraMax M5 Multi-Mode Microplate</td><td>Molecular Devices</td><td>M5</td><td></td></tr><tr><td>Falcon 50 ml Conical Centrifuge Tubes</td><td>Corning, Inc</td><td>&lt;br/&gt; 14-432-22</td><td></td></tr><tr><td>Falcon Cell Strainers 70 &amp;mu;m</td><td>Corning, Inc</td><td>08-771-2</td><td></td></tr><tr><td>Tabletop centrifuge 5430</td><td>Eppendorf</td><td>22620560</td><td></td></tr></tbody></table>

microglia as a surrogate biosensor to determine nanoparticle neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

我们表明，小胶质细胞的功能，作为脑响应于使用上述协议的纳米颗粒的替代传感器。我们的结果包括对下游神经元细胞死亡小胶质细胞活化的毒性作用的测量。 图1展示了协议的工作流程来激活的小胶质细胞，并确定是否分泌的细胞因子减少下丘脑神经元的生存力。 TNF-α的分泌显著增加以下AgNP曝光（ 图2）。当丘脑神经元是从AgNP活化的小胶质?...

Watch this Scientific Journal Video about Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity at JoVE.com

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

小胶质细胞作为替代生物传感器确定纳米颗粒神经毒性

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

microglia-as-surrogate-biosensor-to-determine-nanoparticle

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

6.9K 次观看.  Minneapolis Veterans Affairs Health Care System. Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

视频: Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

注册生物成像，纳米材料可用于诊断和治疗监测

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

科研

生物工程

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.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

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 ...

免疫与感染

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

一个吞噬含量:一个协议，以评估之间的中枢神经系统吞噬细胞和神经元的相互作用

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

神经科学

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.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

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

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

医学

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

原代小胶质的快速和精细的CD11b电磁隔离带增强的纯度和多功能性

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

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.

密度梯度离心法分离小鼠原发胶质细胞的研究

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

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

 用于 CNS 相互作用研究的原代小胶质细胞和神经元共培养方案

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

作者聚焦:用于研究疾病条件下小胶质细胞-神经元相互作用的体外共培养模型

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

小胶质细胞作为替代生物传感器确定纳米颗粒神经毒性

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