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Protocol for Primary Microglial Culture Preparation
原代小胶质细胞培养制备法   

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Abstract

Primary microglia, in either mono-culture or co-culture with neurons or astrocytes, are a powerful tool for studying mechanisms underlying microglial inflammatory responses and cell type-specific interactions in the central nervous system (CNS). This protocol provides the details of how to prepare high purity primary microglia from newborn mouse pups. The overall steps include brain cell dissociation, mixed glial cell culture, and microglia isolation.

Background

In recent years, neuroinflammation has become a hotspot area in neuroscience studies. Inflammatory responses, such as glial activation and cytokine upregulation, were observed in brains of patients with various neurological diseases (Fan et al., 2015; Koshimori et al., 2015; Garden and Campbell, 2016). Neuroinflammation is considered not only a consequence of pathological changes in the brain but also a contributor to disease progression (Schwartz et al., 2013). In addition, the physiological functions of inflammatory pathways, the importance of which were previously underestimated, are being revealed as surprisingly versatile. For instance, activation of the complement signaling pathway is commonly observed in the central nervous system (CNS) in neurological diseases and is suspected to be involved in disease pathophysiology (Michailidou et al., 2015; Loeffler et al., 2008). Now we know that it also plays essential function in the developmental regulation of synaptic refinement (Stevens et al., 2007). Along with the increasing attention on inflammation, interest in microglial function during development, neuroprotection, and pathogenesis continues growing. Microglia are resident innate immune cells of myeloid lineage located in the brain and are critical components of the immune system in the CNS. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. For instance, TREM2 mutations, which affects only microglia, are a genetic risk factor for Alzheimer’s disease (Yuan et al., 2016; Wang et al., 2015). At the same time, developmental roles of microglia are being revealed. For example, synaptic maturation during early development requires microglia and this regulation may underline the pathogenesis of developmental diseases such as autism (Edmonson et al., 2016; Stephan et al., 2012). Tools for studying microglia include in vivo models (e.g., microglia-deficient PU.1 knockout mice [McKercher et al., 1996]) and in vitro systems such as immortalized microglial cell lines and primary microglial culture. While in vivo tools are powerful for demonstrating systematic microglial function, in vitro tools are ideal for mechanistic characterization due to the easy manipulation of experimental factors. Compared to immortalized microglial cell lines, primary microglia better mimic in vivo microglial properties (Stansley et al., 2012). The altered gene expression upon stimulation may be better presented in primary microglia than in microglial cell lines (Stansley et al., 2012; Henn et al., 2009). Here we described a protocol for establishing high purity primary microglial culture derived from neonatal mice and the method has yielded robust results in our work (Lian et al., 2016). Dissociated cells are collected through enzymatic digestion of collected brains and seeded to grow mixed glial culture. Microglia growing on top of a confluent astrocyte layer are purified through mechanical tapping of mixed glial culture.

Materials and Reagents

  1. 15 ml centrifuge tubes (Corning, catalog number: 430052 )
  2. 50 ml centrifuge tubes (Corning, catalog number: 430290 )
  3. 12-well plates (Corning, Costar®, catalog number: 3737 )
  4. New born pups (mouse, P0-P2)
  5. Poly-D-lysine (PDL) (Sigma-Aldrich, catalog number: P6407-5MG )
  6. Ethanol
  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. 10,000 U/ml penicillin-streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  10. Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 24020117 )
  11. 1 M HEPES buffer solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  12. Glucose (Thermo Fisher Scientific, Fisher Scientific, catalog number: D16-3 )
  13. Trypsin, powder (Thermo Fisher Scientific, GibcoTM, catalog number: 27250018 )
  14. Trypsin inhibitor (Sigma-Aldrich, catalog number: T6522-100MG )
  15. Deoxyribonuclease I (DNase I) (Sigma-Aldrich, catalog number: DN25-100MG )
  16. Culture medium (500 ml) (see Recipes)
  17. Dissection medium (500 ml) (see Recipes)
  18. 2.5% trypsin (20 ml) (see Recipes)
  19. 1 mg/ml trypsin inhibitor (20 ml) (see Recipes)
  20. 10 mg/ml DNase (20 ml) (see Recipes)

Equipment

  1. Vented cap T-75 culture flask (Corning, catalog number: 3276 )
  2. Dissection tools
    1. Fine scissors (Fine Science Tools, catalog number: 14060-09 )
    2. Spring scissors (Fine Science Tools, catalog number: 15009-08 )
    3. Curved standard forceps (Fine Science Tools, catalog number: 11052-10 )
    4. Fine forceps (Fine Science Tools, catalog number: 11370-40 )
  3. Centrifuge machine (Eppendorf, model: 5702 )
  4. Hemocytometer
  5. Ventilation hood (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 1323 )
  6. CO2 cell culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 50144906 )
  7. 37 °C water bath (Thermo Fisher Scientific, Thermo ScientificTM, model: TSGP02 )

Procedure

Depending on the experimental design, the number of microglial cells required for experiments varies. Below we list the steps of processing 3 newborn mouse pups to generate mixed glial cultures in two T-75 flasks. In the mixed culture, astrocytes form a confluent cell layer at the bottom and microglia grow on top of the astrocytic layer. The total amount of primary microglia generated from two T-75 flasks should be enough to seed four 12-well plates at a density of 50,000 cells/cm2.

  1. Coat two T-75 culture flasks with 7 ml each of 10 μg/ml PDL for 2 h. Wash the flask bottom with distilled water 3 times before use.
    Note: You can coat more flasks than needed and the unused coated flasks can be stored at 4 °C for months. We usually keep the flasks in plastic wrap to avoid contamination.
  2. Collect new born pups from breeding cages. Keep the pups on a 37 °C heating plate to maintain body temperature. In the meantime, prepare tools and reagents needed for the culture experiment. Spray dissection tools and work space with 75% ethanol. Warm up culture medium (No. 16 in ‘Materials and Reagents’ and No. 1 in ‘Recipes’) in 37 °C water bath.


    Figure 1. Process of newborn mouse pups for primary microglial culture. (a) Use fine scissors to cut off the pup head. (b) Use fine scissors to cut open the scalp along the midline from the posterior end to the middle point between the two eyes. (c) When the thin skull was exposed, put one end of the fine forceps beneath the skull but above the brain tissue and pull toward the snout along the midline so that the brain could then be easily scooped out using curving forceps.

  3. Remove pups from the heating plate. Decapitate and place the heads into a 6 cm Petri dish containing 5 ml cold dissection media (Figure 1a) (No. 17 in ‘Materials and Reagents’ and No. 2 in ‘Recipes’). Use fine scissors to cut open the scalp along the midline starting posteriorly and ending near the snout (Figure 1b). Place one sharp tip of the fine forceps beneath the skull at the posterior end of a brain, and cut the skull by pushing the end from posterior to anterior (Figure 1c). Scoop out the brain using curving forceps and immerse the brains in 5 ml cold dissection media in a new Petri dish.
    Note: Newborn pups have a transparent and soft skull. Using scissors to cut the skull may damage the fragile brain tissue underneath.
  4. Put the Petri dish containing the brains under dissection microscope. Carefully remove the meninges (readers may refer to the video presented by Bowyer et al., 2012 for this step) and collect the cortices and hippocampi. If you use 3 pups, you will get 6 halves. Put 3 halves per Petri dish with 5 ml dissection media and mince the tissue into small pieces using spring scissors.
  5. Transfer the contents of each dish to a 50 ml tube. Wash the dish with dissection media to collect any remaining tissue on the dish and repeat the transfer. Fill the 50 ml tube to reach a final volume of 30 ml dissection media.
  6. Add 1.5 ml 2.5% trypsin to each tube and incubate in the 37 °C water bath for 15 min. Swirl frequently.
  7. Add 1.2 ml 1 mg/ml trypsin inhibitor and incubate for 1 min. Add 750 μl 10 mg/ml DNase to digest the sticky DNA released from dead cells.
  8. Centrifuge the tube at 400 x g for 5 min. Aspirate the supernatant and triturate the pellet with 5 ml warm culture media using a 1 ml pipet tip. Transfer the homogenous cell suspension to a 15 ml tube. If undissociated tissue chunks remain, let them settle and repeat the trituration and transfer step using 3 ml media.
  9. Centrifuge the 15 ml tubes at 400 x g for 5 min. Aspirate the supernatant and resuspend the pellet with 5 ml warmed culture media.
  10. Count the cell density using hemocytometer.
  11. Plate each tube of cells into one coated T-75 flask at the density of 50,000 cells/cm2. Add culture media to reach a volume of 15 ml in the flasks. Put seeded flasks into a CO2 cell culture incubator with 5% CO2, 100% humidity at 37 °C.
  12. Change the culture medium the next day to remove cell debris and then change culture media every 5 days.
  13. In 5-7 days, astrocytes at the bottom of the flask form a confluent cell layer (Figure 2). Microglia and some oligodendrocytes grow on top of the astrocytic layer.


    Figure 2. Astrocytes form a connective confluent layer at the bottom in the mixed glial culture. Arrows point to representative astrocytes. Scale bar = 100 μm.

  14. To collect microglia, vigorously tap the flasks on the bench top and collect the floating cells in conditioned culture media (No need to change media before tapping). The resulting cells are purified microglia. Use a hemocytometer to count the floating cell density and seed the cells at 50,000 cells/cm2 in PDL-coated culture vessels. After 2 h, check that the microglia have attached to the bottom under a microscope. Aspirate medium and replace with fresh culture medium. The microglial cells are ready to use the next day (Figure 3).


    Figure 3. Purified primary microglial culture. Cells were stained with microglial marker protein Iba1. Phase-contrast image was taken to show the cell body. Scale bar = 50 μm.

Data analysis

Getting good quality primary microglial culture is the basis for experiments such as phagocytosis, RNA and protein analysis upon various treatments, and immunocytochemical staining. However, the statistical analysis of experiments after primary microglial culture is beyond the scope of this protocol and therefore, the data analysis process is not discussed here.

Notes

  1. This protocol described microglial preparation from newborn pups (P0-P2). Older pups could also be used since glial cell are not post-mitotic like neurons, but new born pups give better yield.
  2. Most of the procedure should be done in a sterile ventilation hood. Exceptions include the handling of the pups, the brain dissection, centrifugation, and cell counting. Use sterile tubes and dishes. Spray tools and outside surface of tubes and dishes with 70% ethanol before carrying them into the hood. Except for the 37 °C incubations in the water bath and culture incubator, all steps are performed at room temperature and should be completed in a timely manner to enhance viability of cells.
  3. If the mixed glial cells reach confluency but microglial cells are not needed immediately, the mixed glial culture can be passaged. Mixed glial cultures can also be frozen and stored for long term in freezing media composed of DMEM with 20% FBS and 10% DMSO in liquid nitrogen. When the new passage or recovered frozen cells reach confluency, microglia can grow on top of the confluent cell layer and be purified by tapping.
  4. Astrocytes and microglia grow more vigorously than oligodendrocytes in this culture condition. After tapping the mixed glial culture, the collected floating cells may contain some oligodendrocytes. However, microglial cells have much stronger attaching capability than oligodendrocytes. After seeding the floating cells, microglia attach to the culture vessel bottom much more efficiently than oligodendrocytes. At 2 h after seeding before fresh media is added, aspiration of the old media will remove unattached contaminating oligodendrocytes.

Recipes

  1. Culture medium (500 ml)
    450 ml DMEM
    50 ml FBS
    Optionally, you can add 5 ml Pen/Strep
    Filter and store at 4 °C
  2. Dissection medium (500 ml)
    450 ml 1x HBSS
    5 ml 1 M HEPES
    3 g glucose powder
    5 ml Pen/Strep solution
    Filter and store at 4 °C
  3. 2.5% trypsin (20 ml)
    0.5 g trypsin powder dissolved in 20 ml HBSS
    Filter, aliquot, and store at -20 °C
  4. 1 mg/ml trypsin inhibitor (20 ml)
    0.02 g trypsin inhibitor
    Filter, aliquot, and store at -20 °C
  5. 10 mg/ml DNase (20 ml)
    0.2 g DNase I powder dissolved in 20 ml HBSS
    Filter, aliquot, and store at -20 °C

Acknowledgments

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

References

  1. Bowyer, J. F., Thomas, M., Patterson, T. A., George, N. I., Runnells, J. A. and Levi, M. S. (2012). A visual description of the dissection of the cerebral surface vasculature and associated meninges and the choroid plexus from rat brain. J Vis Exp(69): e4285.
  2. Edmonson, C. A., Ziats, M. N. and Rennert, O. M. (2016). A non-inflammatory role for microglia in autism spectrum disorders. Front Neurol 7: 9.
  3. Fan, Z., Okello, A. A., Brooks, D. J. and Edison, P. (2015). Longitudinal influence of microglial activation and amyloid on neuronal function in Alzheimer’s disease. Brain 138(Pt 12): 3685-3698.
  4. Garden, G. A. and Campbell, B. M. (2016). Glial biomarkers in human central nervous system disease. Glia 64(10): 1755-1771.
  5. Henn, A., Lund, S., Hedtjarn, M., Schrattenholz, A., Porzgen, P. and Leist, M. (2009). The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX 26(2): 83-94.
  6. Koshimori, Y., Ko, J. H., Mizrahi, R., Rusjan, P., Mabrouk, R., Jacobs, M. F., Christopher, L., Hamani, C., Lang, A. E., Wilson, A. A., Houle, S. and Strafella, A. P. (2015). Imaging striatal microglial activation in patients with Parkinson’s disease. PLoS One 10(9): e0138721.
  7. Loeffler, D. A., Camp, D. M. and Bennett, D. A. (2008). Plaque complement activation and cognitive loss in Alzheimer’s disease. J Neuroinflammation 5: 9.
  8. 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.
  9. McKercher, S. R., Torbett, B. E., Anderson, K. L., Henkel, G. W., Vestal, D. J., Baribault, H., Klemsz, M., Feeney, A. J., Wu, G. E., Paige, C. J. and Maki, R. A. (1996). Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J 15(20): 5647-5658.
  10. Michailidou, I., Willems, J. G., Kooi, E. J., van Eden, C., Gold, S. M., Geurts, J. J., Baas, F., Huitinga, I. and Ramaglia, V. (2015). Complement C1q-C3-associated synaptic changes in multiple sclerosis hippocampus. Ann Neurol 77(6): 1007-1026.
  11. Schwartz, M., Kipnis, J., Rivest, S. and Prat, A. (2013). How do immune cells support and shape the brain in health, disease, and aging? J Neurosci 33(45): 17587-17596.
  12. Stevens, B., Allen, N. J., Vazquez, L. E., Howell, G. R., Christopherson, K. S., Nouri, N., Micheva, K. D., Mehalow, A. K., Huberman, A. D., Stafford, B., Sher, A., Litke, A. M., Lambris, J. D., Smith, S. J., John, S. W. and Barres, B. A. (2007). The classical complement cascade mediates CNS synapse elimination. Cell 131(6): 1164-1178.
  13. Stephan, A. H., Barres, B. A. and Stevens, B. (2012). The complement system: an unexpected role in synaptic pruning during development and disease. Annu Rev Neurosci 35: 369-389.
  14. Stansley, B., Post, J. and Hensley, K. (2012). A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease. J Neuroinflammation 9: 115.
  15. Wang, Y., Cella, M., Mallinson, K., Ulrich, J. D., Young, K. L., Robinette, M. L., Gilfillan, S., Krishnan, G. M., Sudhakar, S., Zinselmeyer, B. H., Holtzman, D. M., Cirrito, J. R. and Colonna, M. (2015). TREM2 lipid sensing sustains the microglial response in an Alzheimer's disease model. Cell 160(6): 1061-1071.
  16. Yuan, P., Condello, C., Keene, C. D., Wang, Y., Bird, T. D., Paul, S. M., Luo, W., Colonna, M., Baddeley, D. and Grutzendler, J. (2016). TREM2 haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased amyloid compaction and severe axonal dystrophy. Neuron 90(4): 724-739.

简介

单细胞培养或与神经元或星形胶质细胞共培养的原代小胶质细胞是研究中枢神经系统(CNS)中小胶质细胞炎症反应和细胞类型特异性相互作用的机制的有力工具。 该方案提供了如何从新生小鼠幼崽准备高纯度小胶质细胞的细节。 总体步骤包括脑细胞解离,混合胶质细胞培养和小胶质细胞分离。
【背景】近年来,神经炎症已成为神经科学研究的热点领域。在各种神经系统疾病患者的脑中观察到炎症反应,如胶质细胞激活和细胞因子上调(Fan等,2015; Koshimori等,2015; Garden and Campbell,2016)。神经炎症不仅被认为是脑内病理变化的结果,而且也是疾病进展的贡献者(Schwartz等,2013)。此外,炎症途径的生理功能,其重要性以前被低估,正在被揭示为令人惊讶的多才多艺。例如,补体信号通路的激活通常在神经系统疾病中的中枢神经系统(CNS)中被观察到,并被怀疑参与疾病病理生理学(Michailidou等人,2015; Loeffler等人,2008)。现在我们知道它也在突触细化发育调控中发挥重要作用(Stevens et al。,2007)。随着对炎症的关注越来越多,开发期间对小胶质细胞功能的兴趣,神经保护和发病机制持续增长。小胶质细胞是位于脑中的骨髓谱系的常见先天免疫细胞,是CNS中免疫系统的关键组分。某些神经系统疾病中小胶质细胞的激活可能直接参与致病过程。例如,仅影响小神经胶质细胞的TREM2突变是阿尔茨海默病的遗传风险因素(Yuan et al。,2016; Wang et al。,2015)。同时,小胶质细胞的发育作用正在被揭示。例如,早期发育期间的突触成熟需要小胶质细胞,这种调节可能会强调发育性疾病如自闭症的发病机制(Edmonson等,2016; Stephan等,2012)。用于研究小胶质细胞的工具包括体内模型(例如小胶质细胞缺陷型PU.1敲除小鼠[McKercher等,1996])和体外系统,例如永生化小胶质细胞系和原代小胶质细胞培养。虽然体内工具对于证明系统性小胶质细胞功能是强大的,但由于易于操作实验因素,体外工具是机械表征的理想选择。与永生化小胶质细胞系相比,原代小胶质细胞更好地模拟体内小胶质细胞性质(Stansley等,2012)。刺激后改变的基因表达可能在原代小胶质细胞中比在小胶质细胞系中更好地呈现(Stansley等人,2012; Henn等人,2009)。在这里,我们描述了一种建立来自新生小鼠的高纯度原代小胶质细胞培养的方案,该方法在我们的工作中产生了强大的结果(Lian et al。,2016)。通过酶解消化收集的大脑收集解离的细胞并接种培养混合胶质细胞。在融合的星形胶质细胞层顶部生长的小胶质细胞通过混合神经胶质培养物的机械攻丝来纯化。

材料和试剂

  1. 15ml离心管(Corning,目录号:430052)
  2. 50ml离心管(Corning,目录号:430290)
  3. 12孔板(Corning,Costar ,目录号:3737)
  4. 新生小狗(老鼠,P0-P2)
  5. 聚-D-赖氨酸(PDL)(Sigma-Aldrich,目录号:P6407-5MG)
  6. 乙醇
  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. 10,000 U/ml青霉素 - 链霉素(Pen/Strep)(Thermo Fisher Scientific,Gibco< sup>,目录号:15140122)
  10. Hanks平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM ,目录号:24020117)
  11. 1 M HEPES缓冲溶液(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
  12. 葡萄糖(Thermo Fisher Scientific,Fisher Scientific,目录号:D16-3)
  13. 胰蛋白酶,粉末(Thermo Fisher Scientific,Gibco TM ,目录号:27250018)
  14. 胰蛋白酶抑制剂(Sigma-Aldrich,目录号:T6522-100MG)
  15. 脱氧核糖核酸酶I(DNase I)(Sigma-Aldrich,目录号:DN25-100MG)
  16. 培养基(500ml)(参见配方)
  17. 解剖介质(500ml)(见配方)
  18. 2.5%胰蛋白酶(20ml)(参见配方)
  19. 1mg/ml胰蛋白酶抑制剂(20ml)(见Recipes)
  20. 10mg/ml脱氧核糖核酸酶(20ml)(见配方)

设备

  1. 通气盖T-75培养瓶(Corning,目录号:3276)
  2. 解剖工具
    1. 精细剪刀(Fine Science Tools,目录号:14060-09)
    2. 弹簧剪(Fine Science Tools,目录号:15009-08)
    3. 弯曲标准镊子(Fine Science Tools,目录号:11052-10)
    4. 精细镊子(Fine Science Tools,目录号:11370-40)
  3. 离心机(Eppendorf,型号:5702)
  4. 血细胞计数器
  5. 通风罩(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:1323)
  6. CO 2细胞培养孵育器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:50144906)
  7. 37℃水浴(Thermo Fisher Scientific,Thermo Scientific ,型号:TSGP02)

程序

根据实验设计,实验所需的小胶质细胞的数量不同。下面我们列出了处理3只新生小鼠幼仔以在两个T-75瓶中产生混合胶质培养物的步骤。在混合培养中,星形胶质细胞在底部形成汇合的细胞层,小胶质细胞在星形胶质层的顶部生长。从两个T-75烧瓶产生的原代小胶质细胞的总量应足以以50,000细胞/cm 2的密度接种四个12孔板。

  1. 涂覆两个T-75培养瓶与7毫升每个10微克/毫升PDL 2小时。使用前用蒸馏水洗涤底部3次。
    注意:您可以涂覆比所需更多的烧瓶,未使用的涂覆烧瓶可以在4°C储存数个月。我们通常保持烧瓶在塑料包装,以避免污染。
  2. 从繁殖笼收集新出生的幼犬。保持小狗在37°C加热板,以保持体温。同时,准备培养实验所需的工具和试剂。喷洒清扫工具和工作空间与75%乙醇。在37℃水浴中预热培养基("材料和试剂"中的第16号和"配方"中的第1号)。


    图1.用于原代小胶质细胞培养的新生小鼠幼崽的过程。(a)使用细剪刀剪断幼崽头。 (b)用细剪刀沿着中线从后端到两眼之间的中点切开头皮。 (c)当薄的头骨暴露时,将细镊子的一端放在头骨下面,但在脑组织上面,沿着中线拉向鼻涕,使得大脑可以很容易地用弯曲镊子舀出。 >
  3. 从加热板上取下幼崽。断头并将头放入包含5ml冷解剖介质(图1a)("材料和试剂"中的No.17和"Recipes"中的No.2)的6cm培养皿中。使用精细剪刀沿着中线开始头皮,从后面开始,并在鼻子附近结束(图1b)。将精细镊子的一个锋利的尖端在头骨后面的大脑的后端,并通过将末端从后面推到前面切开颅骨(图1c)。使用弯曲镊子舀出大脑,将大脑浸入新的培养皿中的5ml冷的解剖培养基中。
    注意:新生小狗有一个透明和软的头骨。使用剪刀剪下颅骨可能会损坏下面脆弱的脑组织。
  4. 将含有大脑的培养皿放在解剖显微镜下。小心地去除脑膜(读者可以参考由Bowyer等人提出的视频。,2012为这一步),并收集皮质和海马。如果你使用3只小狗,你会得到6个半。放置3个半培养皿与5毫升解剖介质,并使用弹簧剪刀将组织切成小块。
  5. 将每个碟的内容物转移到50ml管中。用解剖培养基洗涤培养皿,收集培养皿上剩余的任何组织,并重复转移。填充50ml管,达到最终体积为30毫升解剖介质。
  6. 向每个管中加入1.5ml 2.5%胰蛋白酶,并在37℃水浴中孵育15分钟。经常旋转。
  7. 加入1.2ml 1mg/ml胰蛋白酶抑制剂并孵育1分钟。加入750微升10毫克/毫升DNA酶消化从死细胞释放的粘性DNA
  8. 在400×g离心管5分钟。吸出上清液,用5ml温热的培养基,使用1ml移液管尖端研磨沉淀。转移均匀细胞悬浮液到15毫升管。如果未解离的组织块残留,让他们沉淀,并使用3ml培养基重复研磨和转移步骤。
  9. 以400×g离心15ml管5分钟。吸出上清液,并用5ml温热的培养基重悬沉淀。
  10. 使用血细胞计数器计数细胞密度
  11. 将每个管的细胞以50,000细胞/cm 2的密度铺在一个涂覆的T-75烧瓶中。加入培养基,以在烧瓶中达到15ml的体积。将接种的培养瓶放入具有5%CO 2,100%湿度的CO 2细胞培养箱中,37℃。
  12. 第二天更换培养基以去除细胞碎片,然后每5天更换培养基
  13. 在5-7天,在烧瓶底部的星形胶质细胞形成融合细胞层(图2)。小胶质细胞和一些少突胶质细胞生长在星形胶质层的顶部。


    图2.星形胶质细胞在混合胶质培养物的底部形成连接融合层。箭头指向代表性星形胶质细胞。比例尺=100μm。

  14. 收集小胶质细胞,大力点击在台面上的烧瓶,收集浮动细胞在条件培养基(在开始之前不需要更换介质)。所得细胞是纯化的小胶质细胞。使用血细胞计数器计数浮动细胞密度和种子细胞在50,000细胞/cm 2在PDL包被培养容器中。 2小时后,检查小胶质细胞在显微镜下附着在底部。吸出培养基并更换新鲜培养基。小胶质细胞可以在第二天使用(图3)。


    图3.纯化的原代小胶质细胞培养物。细胞用小胶质细胞标记蛋白Iba1染色。获取相位对比图像以显示细胞体。比例尺=50μm。

数据分析

获得良好质量的原代小胶质细胞培养是实验的基础,例如吞噬作用,各种处理的RNA和蛋白质分析以及免疫细胞化学染色。然而,初级小胶质细胞培养后的实验的统计分析超出了本协议的范围,因此,数据分析过程在这里不讨论。

笔记

  1. 该协议描述了从新生小鼠(P0-P2)的小胶质细胞制备。也可以使用较老的幼崽,因为神经胶质细胞不是有丝分裂后的神经元,但新生的幼崽产生更好的产量。
  2. 大多数程序应在无菌通风罩中进行。例外包括幼仔的处理,脑解剖,离心和细胞计数。使用无菌管和餐具。喷雾工具和管子和菜肴的外表面与70%乙醇,在他们进入敞篷前。除了在水浴和培养孵化器中37℃孵育之外,所有步骤在室温下进行,并且应当及时完成以增强细胞的活力。
  3. 如果混合的神经胶质细胞达到融合,但不需要立即使用小胶质细胞,则可以传代混合的神经胶质培养物。混合的胶质细胞培养物也可以冷冻并长期储存在由在液氮中含有20%FBS和10%DMSO的DMEM组成的冷冻培养基中。当新的传代或回收的冷冻细胞达到汇合时,小神经胶质细胞可以在融合细胞层的顶部生长并通过敲打来纯化。
  4. 在该培养条件下,星形胶质细胞和小神经胶质细胞比少突胶质细胞生长更强。在轻拍混合胶质细胞培养物后,收集的浮动细胞可能含有一些少突胶质细胞。然而,小胶质细胞比少突胶质细胞具有更强的附着能力。在接种浮细胞后,小胶质细胞比少突胶质细胞更有效地附着于培养容器底部。在接种后2小时,在加入新鲜培养基之前,吸去旧培养基将除去未附着的污染少突胶质细胞。

食谱

  1. 培养基(500ml)
    450 ml DMEM
    50ml FBS
    或者,您可以添加5毫升Pen/Strep
    过滤并储存在4°C
  2. 解剖介质(500ml)
    450 ml 1x HBSS
    5 ml 1 M HEPES
    3 g葡萄糖粉
    5ml Pen/Strep溶液
    过滤并储存在4°C
  3. 2.5%胰蛋白酶(20ml) 0.5克胰蛋白酶粉溶解在20ml HBSS中 过滤,等分,并储存在-20°C
  4. 1mg/ml胰蛋白酶抑制剂(20ml) 0.02克胰蛋白酶抑制剂
    过滤,等分,并储存在-20°C
  5. 10mg/ml DNA酶(20ml) 0.2g DNase I粉末溶于20ml HBSS中 过滤,分装,并在-20°C下保存

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

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  2. Edmonson,CA,Ziats,MN和Rennert,OM(2016)。  小胶质细胞在自闭症谱系障碍中的非炎性作用。前neurol 7:9.
  3. Fan,Z.,Okello,AA,Brooks,DJ和Edison,P.(2015)。  小胶质细胞活化和淀粉样蛋白对阿尔茨海默病中神经元功能的纵向影响。脑 138(Pt 12):3685-3698。 >
  4. Garden,GA和Campbell,BM(2016)。  Glial 人类中枢神经系统疾病中的生物标志物。 Glia 64(10):1755-1771。
  5. Henn,A.,Lund,S.,Hedtjarn,M.,Schrattenholz,A.,Porzgen,P.和Leist,M。(2009)。  BV2细胞作为原代小胶质细胞培养物的替代模型系统或用于检查脑炎症的动物实验的适合性。 ALTEX 26(2):83-94。
  6. Koshimori,Y.,Ko,JH,Mizrahi,R.,Rusjan,P.,Mabrouk,R.,Jacobs,MF,Christopher,L.,Hamani,C.,Lang,AE,Wilson,AA,Houle,和Strafella,AP(2015)。  成像纹状体小胶质细胞激活在帕金森病患者中。 PLoS One 10(9):e0138721。
  7. Loeffler,DA,Camp,DM and Bennett,DA(2008)。  斑块补体激活和阿尔茨海默病中的认知缺失 5:9.
  8. 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学 J Neurosci 36(2):577-589。
  9. McKercher,SR,Torbett,BE,Anderson,KL,Henkel,GW,Vestal,DJ,Baribault,H.,Klemsz,M.,Feeney,AJ,Wu,??GE,Paige,CJand Maki,RA(1996) ; PU.1基因的靶向破坏导致多种造血异常。 EMBO J 15(20):5647-5658
  10. Michaelidou,I.,Willems,JG,Kooi,EJ,van Eden,C.,Gold,SM,Geurts,JJ,Baas,F.,Huitinga,I。和Ramaglia, ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25727254"target ="_ blank">多发性硬化海马中补体C1q-C3相关的突触变化。 Ann Neurol 77(6):1007-1026。
  11. Schwartz,M.,Kipnis,J.,Rivest,S.和Prat,A.(2013)。  免疫细胞如何支持健康,疾病和衰老的大脑? J Neurosci 33(45):17587-17596。
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  16. 这些研究结果表明,这些研究结果表明,该方法能够有效地抑制细胞凋亡。   小鼠和人类中的TREM2单倍体缺失会损害小胶质细胞屏障功能减少淀粉样蛋白紧缩和严重轴突营养不良。 Neuron 90(4):724-739。
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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). Protocol for Primary Microglial Culture Preparation. Bio-protocol 6(21): e1989. DOI: 10.21769/BioProtoc.1989.
  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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