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Microglial Phagocytosis Assay
小胶质细胞吞噬作用试验   

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Abstract

Phagocytosis is essential for microglial clearance of apoptotic cells, extracellular protein aggregates, and infectious bacteria in the central nervous system (CNS). While the preparation of primary microglial culture has been described elsewhere, this protocol describes the microglial phagocytosis experimental procedure and the subsequent measurement of microglial phagocytic ability using fluorescent latex beads or fluorescent amyloid beta 42 (Aβ42) peptides.

Background

Microglia play multiple roles in the central nervous system (CNS). Upon stimulation, microglia present complicated inflammatory responses including altered gene expression and morphological changes (Heneka et al., 2014; Cunningham, 2013). Cytokines are a critical cluster of proteins among the list of altered expression molecules by activated microglia. Through the potent signaling-capable cytokine receptors expressed on astrocytes, neurons, and other brain cell types, microglia communicate, recruit, and coordinate inflammatory events (Smith et al., 2012). Besides cytokine secretion, phagocytosis, which involves morphological changes in microglia, also adds to their role as guardians of environmental homeostasis within the CNS milieu. Microglial phagocytosis of pathogens, extracellular protein aggregates, and apoptotic cell debris dampens inflammation and protects neurons (Fu et al., 2014). Apart from pathogenic conditions, microglial phagocytosis is also involved in CNS development and synaptogenesis through eliminating nonfunctional synapses. Deficient or excess microglial phagocytic ability could lead to abnormal synaptic connections and deposits of aggregated proteins (Schafer et al., 2012; Lian et al., 2016). Here we describe a protocol for measuring microglial phagocytic ability using in vitro cultured primary microglia. To mimic exogenous particles and protein aggregates, we used latex beads and amyloid β protein as the substrates for microglia to engulf.

Materials and Reagents

  1. 24-well plates (Corning, Costar®, catalog number: 3527 )
  2. 12 mm glass coverslips (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-545-81 )
  3. 1.5 ml centrifuge tubes (Corning, Axygen®, catalog number: MCT-150-R )
  4. Fluorescent latex beads of 1 μm diameter (Sigma-Aldrich, catalog number: L1030 )
  5. Poly-D-lysine (PDL) (Sigma-Aldrich, catalog number: P6407-5MG )
  6. Distilled water (Thermo Fisher Scientific, GibcoTM, catalog number: 15230147 )
  7. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11995065 )
  8. Fetal bovine serum (FBS) (GE Healthcare, HycloneTM, catalog number: SH30088.03 )
  9. FAM-labelled Aβ42 peptides (AnaSpec, catalog number: AS-23526-01 )
  10. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
  11. Phosphate buffered salt (PBS)
  12. 4% PFA (Santa Cruz Biotechnology, catalog number: sc-281692 )
  13. Mounting medium with DAPI (Vector Laborstories, catalog number: H-1200 )
  14. Microglial culture media (500 ml) (see Recipes)

Equipment

  1. Ventilation hood (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 1323 )
  2. CO2 cell culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 50144906 )
  3. 37 °C water bath (Thermo Fisher Scientific, Thermo ScientificTM, model: TSGP02 )
  4. Cell counter

Software

  1. ImageJ (http://fiji.sc/)

Procedure

  1. Coat coverslips with 10 μg/ml PDL (250 μl/well for a 24-well plate well) for 2 h at room temperature. Wash the coverslips with distilled water 3 times and aspirate the water before use.
    Note: Extra coated plates could be store at 4 °C for months.
  2. Prepare purified primary microglial cells. Seed microglia onto coverslips at a density of 50,000 cells/cm2. Put the cultures into an incubator containing 5% CO2 and 100% humidity at 37 °C. Microglia attach to the wells within 2 h of seeding. Replenish the wells with fresh, pre-warmed microglial culture medium (No. 14 in ‘Materials and Reagents’ and No. 1 in ‘Recipes’) after cells are attached. Replace cultures into the incubator.
  3. Allow 24 h for the microglial cells to recover, after which the cells will be ready for the phagocytosis assay the following day.
  4. If using fluorescent beads:
    Pre-opsonize aqueous green fluorescent latex beads in FBS for 1 h at 37 °C. The ratio of beads to FBS is 1:5. Dilute the bead-containing FBS with DMEM to reach the final concentrations for beads and FBS in DMEM of 0.01% (v/v) and 0.05% (v/v), respectively.
    If using fluorescent Aβ42:
    Prepare the fluorescent Aβ42 stock solution according to the manufacturer’s recommendations. We dissolve the peptide in DMSO to obtain a 0.1 mM stock (200x). Dilute the reconstituted Aβ42 peptides in DMEM to reach a final concentration of 500 nM and incubate the solution at 37 °C for 1 h to promote Aβ42 aggregation.
  5. Replace microglial conditioned culture media with beads- or Aβ-containing DMEM and incubate cultures at 37 °C for 1 h. For a well of a 24-well plate, we add 250 μl beads- or Aβ-containing DMEM.
  6. Wash cultures thoroughly with ice-cold PBS 5 times and then fix the cells using 4% PFA for 15 min.
  7. Perform immunohistochemistry for microglial proteins which can mark cell shape and counterstain the culture with DAPI. We visualize green fluorescent beads and FAM-Aβ with the green channel and use the red channel for Iba1 staining (Figures 1 and 2).
    Note: Iba1 works well in our hands. It not only demonstrates cell morphology but also excludes other cell types since it is a microglial-specific marker. You should use a secondary antibody detectable by a different channel than the fluorescent beads or Aβ.
  8. Image the microglial culture using a confocal microscope. For latex beads, we recommend imaging at low to medium magnification (10x or 20x) so that more cells can be imaged in one field. For Aβ42, we recommend imaging at medium or high magnification (e.g., 20x or 40x) to maintain sufficient resolution for visualizing the Aβ signal.


    Figure 1. Microglial phagocytosis assay using fluorescent latex beads. White arrowheads point to phagocytic microglial cells containing beads inside the cell body. Scale bar = 50 μm.


    Figure 2. Microglial phagocytosis assay using fluorescent Aβ42. Scale bar = 50 μm.

Data analysis

Below we use the example of images of green fluorescent beads or Aβ and red Iba1 staining signal to explain the data processing procedure.
Open the file with ImageJ (We recommend the Fiji version of ImageJ. It has higher compatibility with many file formats generated by different microscope systems and is loaded with a variety of useful plugins).

  1. For microglial phagocytosis of latex beads
    1. Count the total cell number in each field. You can use the cell counter ImageJ plugin to count cells manually or count the total number of cell nuclei by using the ‘Analyze particles’ tool to automatically identify DAPI-positive areas after threshold adjustment (Ntotal) (Figure 3).
    2. Make a composite image of Iba1 and bead signals, and manually count cells having beads inside the cell body (Ncell with beads) using the cell counter plugin (Figure 4).
    3. Percentage of phagocytic cells is determined as: Ncell with beads/Ntotal.



      Figure 3. Counting the total cell number in a field. In the DAPI channel, adjust the color threshold until all the nuclei are highlighted. Select ‘Analyze particles’ in the menu, define the particle size from 20 μm2 to infinity (20 μm2 is usually large enough to exclude non-specific signals) and check the options of ‘Exclude on edges’ and ‘Summarize’. The output window will show the total number of cells counted.


      Figure 4. Counting phagocytic cells using the cell counter plugin. Make a composite image of beads and Iba1, open the cell counter plugin, initialize the image, chose a cell type, and click on the cells having beads inside the cell body. The number of selected cells will show up in the plugin window.


      Figure 5. Identify microglial cell body. Open the Iba1 image, adjust the color threshold to highlight the cell body, and define the particle size. Check the option of adding the positive areas to the ROI manager (‘Add to Manager’) and the areas will be numbered and listed in the ROI Manager.

  2. For phagocytosis assays using Aβ42
    1. Identify individual microglial cells as regions of interest (ROIs) using the Analyze particles function and the ROI manager in the Iba1 channel (Figure 5).
    2. Exclude ROIs that represent cell debris (check the DAPI channel and exclude the ROIs without a nucleus) or have large FAM-labelled inclusions of Aβ aggregates, as these artefacts will introduce large variance into your measurement (check the Aβ channel and exclude the ROIs with bulky Aβ) (Figure 6).
    3. Switch to the Aβ channel. Adjust the color threshold to highlight Aβ-positive regions and measure both the fluorescence intensity and percentage area of Aβ inside each ROI (Figure 7).


      Figure 6. Optimize the selection of positive areas. Switch to the DAPI channel and exclude the regions without nucleus (yellow arrowheads). Switch to the Aβ channel and delete the cells which engulf large Aβ aggregates (white arrowheads).


      Figure 7. Aβ measurement. In the Aβ channel, adjust the color threshold to select Aβ positive signals, select all the regions in the ROI manager, and measure the mean grey value and area fraction of the Aβ signals in the ROIs.

Recipes

  1. Microglial culture media (500 ml)
    Add 50 ml FBS to 450 ml DMEM for a final serum concentration of 10%

Acknowledgments

We thank the R01s from the NIH (AG032051, AG020670, and NS076117 to H.Z.) for supporting this work.

References

  1. Cunningham, C. (2013). Microglia and neurodegeneration: the role of systemic inflammation. Glia 61(1): 71-90.
  2. Fu, R., Shen, Q., Xu, P., Luo, J. J. and Tang, Y. (2014). Phagocytosis of microglia in the central nervous system diseases. Mol Neurobiol 49(3): 1422-1434.
  3. Heneka, M. T., Kummer, M. P. and Latz, E. (2014). Innate immune activation in neurodegenerative disease. Nat Rev Immunol 14(7): 463-477.
  4. Lian, H., Litvinchuk, A., Chiang, A. C., Aithmitti, N., Jankowsky, J. L. and Zheng, H. (2016). Astrocyte-microglia cross talk through complement activation modulates amyloid pathology in mouse models of Alzheimer's disease. J Neurosci 36(2): 577-589.
  5. Schafer, D. P., Lehrman, E. K., Kautzman, A. G., Koyama, R., Mardinly, A. R., Yamasaki, R., Ransohoff, R. M., Greenberg, M. E., Barres, B. A. and Stevens, B. (2012). Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron 74(4): 691-705.
  6. Smith, J. A., Das, A., Ray, S. K. and Banik, N. L. (2012). Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Res Bull 87(1): 10-20.

简介

吞噬作用对于中枢神经系统(CNS)中细胞凋亡细胞,细胞外蛋白质聚集体和感染性细菌的小胶质细胞清除至关重要。 虽然其他地方已经描述了原代小胶质细胞培养物的制备,但是该方案描述了小胶质细胞吞噬实验程序和随后使用荧光乳胶珠或荧光淀粉样蛋白β42(Aβ42)肽测量小胶质细胞吞噬能力。
【背景】小神经胶质细胞在中枢神经系统(CNS)中起多重作用。刺激后,小胶质细胞呈现复杂的炎症反应,包括改变的基因表达和形态变化(Heneka等,2014; Cunningham,2013)。细胞因子是通过活化的小胶质细胞改变的表达分子列表中的关键的蛋白质簇。通过在星形胶质细胞,神经元和其他脑细胞类型上表达的有效的信号传导能力的细胞因子受体,小胶质细胞传达,募集和协调炎性事件(Smith等,2012)。除了细胞因子分泌,吞噬细胞,其涉及小胶质细胞的形态学变化,也增加了他们作为CNS环境中环境稳态监护人的作用。病原体的小胶质细胞吞噬作用,细胞外蛋白质聚集体和凋亡细胞碎片抑制炎症和保护神经元(Fu et al。,2014)。除致病条件外,小神经胶质细胞吞噬也通过消除非功能性突触参与CNS发育和突触发生。缺乏或过量的小神经胶质细胞吞噬能力可导致突触连接异常和聚集蛋白沉积(Schafer et al。,2012; Lian et al。,2016)。这里我们描述一种使用体外培养的原代小胶质细胞测量小胶质细胞吞噬能力的方案。为了模拟外源性颗粒和蛋白质聚集体,我们使用乳胶珠和淀粉样蛋白β蛋白作为小胶质细胞吞噬的底物。

材料和试剂

  1. 24孔板(Corning,Costar ,目录号:3527)
  2. 12mm玻璃盖玻片(Thermo Fisher Scientific,Fisher Scientific,目录号:12-545-81)
  3. 1.5ml离心管(Corning,Axygen ,目录号:MCT-150-R)
  4. 1μm直径的荧光乳胶珠(Sigma-Aldrich,目录号:L1030)
  5. 聚-D-赖氨酸(PDL)(Sigma-Aldrich,目录号:P6407-5MG)
  6. 蒸馏水(Thermo Fisher Scientific,Gibco TM ,目录号:15230147)
  7. Dulbecco's改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM ,目录号:11995065)
  8. 胎牛血清(FBS)(GE Healthcare,Hyclone ,目录号:SH30088.03)
  9. FAM标记的Aβ42肽(AnaSpec,目录号:AS-23526-01)
  10. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:472301)
  11. 磷酸盐缓冲盐(PBS)
  12. 4%PFA(Santa Cruz Biotechnology,目录号:sc-281692)
  13. 使用DAPI(Vector Laborstories,目录号:H-1200)的固定介质
  14. 小胶质细胞培养基(500ml)(见配方)

设备

  1. 通风罩(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:1323)
  2. CO 2细胞培养孵育器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:50144906)
  3. 37℃水浴(Thermo Fisher Scientific,Thermo Scientific TM ,型号:TSGP02)
  4. 单元格计数器

软件

  1. ImageJ( http://fiji.sc/

程序

  1. 用10μg/ml PDL(对于24孔板孔,250μl/孔)在室温下涂覆盖玻片2小时。用蒸馏水洗涤盖玻片3次,并在使用前抽吸水。
    注意:额外的涂布板可以在4℃下储存数月。
  2. 准备纯化的原代小胶质细胞。以50,000细胞/cm 2的密度将种小胶质细胞接种到盖玻片上。将培养物放入含有5%CO 2和100%湿度的培养箱中,37℃。小胶质细胞在接种的2小时内附着到孔上。在细胞附着后,用新鲜的,预热的小胶质细胞培养基("材料和试剂"中的第14号和"Recipes"中的第1号)补充孔。将培养物更换为培养箱。
  3. 允许24小时小胶质细胞恢复,之后细胞将准备好吞噬测定第二天。
  4. 如果使用荧光珠:
    在37℃下将水性绿色荧光乳胶珠在FBS中预先调理1小时。珠与FBS的比例为1:5。用DMEM稀释含珠的FBS以分别在0.01%(v/v)和0.05%(v/v)的DMEM中达到珠和FBS的最终浓度。
    如果使用荧光Aβ42:
    根据制造商的建议制备荧光Aβ42储备溶液。我们将肽溶解在DMSO中以获得0.1mM储备液(200x)。稀释重组Aβ42肽在DMEM中达到终浓度500 nM,并在37℃孵育1小时,以促进Aβ42聚集的溶液。
  5. 替换小胶质条件培养基与珠或含有Aβ的DMEM,并孵育培养物在37℃下1小时。对于24孔板的孔,我们加入250μl含珠子或Aβ的DMEM
  6. 用冰冷PBS彻底洗涤培养物5次,然后用4%PFA固定细胞15分钟。
  7. 执行小胶质蛋白的免疫组织化学,可以标记细胞形状和用DAPI复染培养物。我们用绿色通道显示绿色荧光珠和FAM-Aβ,并使用红色通道进行Iba1染色(图1和图2)。
    注意:Iba1在我们手中工作得很好。它不仅显示细胞形态,而且排除其他细胞类型,因为它是小胶质细胞特异性标记。您应使用可通过与荧光珠或Aβ不同的通道检测的二抗。
  8. 使用共聚焦显微镜图像小胶质细胞培养。对于乳胶珠,我们建议在低至中等放大倍数(10x或20x)成像,以便更多的细胞可以在一个领域成像。对于Aβ42,我们建议在中等或高放大倍率(例如,20x或40x)成像以保持足够的分辨率以显现Aβ信号。


    图1.使用荧光乳胶珠的小胶质细胞吞噬测定。白色箭头指向在细胞体内含有珠的吞噬小胶质细胞。比例尺=50μm。


    图2.使用荧光Aβ42的小胶质细胞吞噬测定。比例尺=50μm。

数据分析

下面我们以绿色荧光珠或Aβ和红色Iba1染色信号的图像为例来说明数据处理程序。
使用ImageJ打开文件(我们推荐Fiji版本的ImageJ,它与许多不同显微镜系统生成的文件格式具有更高的兼容性,并且加载了各种有用的插件)。

  1. 用于乳胶珠的小胶质细胞吞噬
    1. 计算每个字段中的总细胞数。您可以使用细胞计数器ImageJ插件手动计数细胞或计数细胞核的总数,通过使用"分析粒子"工具自动识别阈值调整后的DAPI阳性区域(N )图3)。
    2. 制作Iba1和珠信号的复合图像,并使用细胞计数器插件手动计数细胞体内具有珠的细胞( Ncell with beads )(图4)。
    3. 吞噬细胞的百分比确定为:具有珠子/N总计的N细胞。



      图3.计算字段中的总细胞数。在DAPI通道中,调整颜色阈值,直到突出显示所有核。选择菜单中的"分析粒子",定义从20μm 2到无穷大(20μm 2 通常大到足以排除非特异性信号)的粒径,并检查'在边上排除'和'总结'的选项。输出窗口将显示计数的细胞总数。


      图4.使用细胞计数器插件计数吞噬细胞。制作珠子和Iba1的复合图像,打开细胞计数器插件,初始化图像,选择一种细胞类型,然后单击具有珠子的细胞在细胞体内。所选单元格的数量将显示在插件窗口中。


      图5.识别小胶质细胞体。打开Iba1图像,调整颜色阈值以突出显示细胞体,并定义粒径。检查向ROI管理器添加正面区域的选项("添加到经理"),并且区域将被编号并在ROI管理器中列出。

  2. 用于使用Aβ42的吞噬测定
    1. 使用分析粒子功能和Iba1通道中的ROI经理来识别单个小胶质细胞作为感兴趣区域(ROI)(图5)。
    2. 排除代表细胞碎片的ROI(检查DAPI通道并排除没有核的ROI)或具有大的FAM标记的Aβ聚集体包涵体,因为这些假象会在测量中引入大的方差(检查Aβ通道并排除ROI大体积Aβ)(图6)
    3. 切换到Aβ通道。调整颜色阈值以突出Aβ阳性区域,并测量每个ROI内的Aβ的荧光强度和百分比面积(图7)。


      图6.优化正面区域的选择。切换到DAPI通道并排除没有核心的区域(黄色箭头)。切换到Aβ通道,并删除吞噬大Aβ聚集体(白色箭头)的细胞

      图7.Aβ测量。在Aβ通道中,调整颜色阈值以选择Aβ阳性信号,选择ROI管理器中的所有区域,并测量Aβ信号的平均灰度值和面积分数在ROI。

食谱

  1. 小胶质细胞培养基(500ml)
    加入50ml FBS到450ml DMEM中,最终血清浓度为10%

致谢

我们感谢来自NIH(AG032051,AG020670和NS076117到H.Z.)的R01s支持这项工作。

参考文献

  1. Cunningham,C。(2013)。  小胶质细胞和神经变性:全身炎症的作用。 61(1):71-90
  2. m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m .nih.gov/pubmed/24395130"target ="_ blank">中枢神经系统疾病中小胶质细胞的吞噬作用 Mol Neurobiol 49(3):1422-1434。 >
  3. Heneka,MT,Kummer,MP和Latz,E。(2014)。  先天免疫激活在神经变性疾病中。 Nat Rev Immunol 14(7):463-477。
  4. Lian,H.,Litvinchuk,A.,Chiang,AC,Aithmitti,N.,Jankowsky,JL和Zheng,H。(2016)。  通过补体激活的星形胶质细胞 - 小胶质细胞交谈可调节阿尔茨海默氏病小鼠模型中的淀粉样蛋白病理。 36(2):577-589。
  5. Schafer,DP,Lehrman,EK,Kautzman,AG,Koyama,R.,Mardinly,AR,Yamasaki,R.,Ransohoff,RM,Greenberg,ME,Barres,BAand Stevens,B。(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22632727"target ="_ blank">小胶质细胞以活性和补体依赖性方式塑造出生后神经回路。 a>  神经元  74(4):691-705。
  6. Smith,JA,Das,A.,Ray,SK和Banik,NL(2012)。  从小胶质细胞释放的促炎细胞因子在神经变性疾病中的作用。 Brain Res Bull 87(1):10-20。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Lian, H., Roy, E. and Zheng, H. (2016). Microglial Phagocytosis Assay. Bio-protocol 6(21): e1988. DOI: 10.21769/BioProtoc.1988.
  2. Lian, H., Litvinchuk, A., Chiang, A. C., Aithmitti, N., Jankowsky, J. L. and Zheng, H. (2016). Astrocyte-microglia cross talk through complement activation modulates amyloid pathology in mouse models of Alzheimer's disease. J Neurosci 36(2): 577-589.
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