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Clearance of dead brain tissue including the dead neurons through phagocytosis is an endogenous function of microglia in the brain, which is critical for inflammation resolution after ischemic stroke or head trauma. By regulating the function or polarization status of microglia, we may control their phagocytosis efficacy and therefore the cleanup process for the dead brain tissue. We cultured rat cortical neurons and microglia from the same litter of embryos. The cultured neurons are subjected to irradiation for inducing neuronal apoptosis. After labeling with propidium iodide (PI), the dead neurons (DNs) are exposed to the cultured microglia for phagocytosis assay. By counting the number of DNs in each microglia, we calculate the phagocytosis index to quantify the phagocytosis efficacy of microglia toward DNs. The protocol is divided into 4 sections: A) culturing rat cortical neurons from pre-natal rat embryos, B) preparing dead neurons as phagocytosis target, C) culturing rat brain microglia, D) quantifying phagocytosis index of microglia toward the dead neurons.

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Phagocytosis Assay of Microglia for Dead Neurons in Primary Rat Brain Cell Cultures
原代大鼠脑细胞培养物中小神经胶质对死亡神经元吞噬作用的测定

神经科学 > 细胞机理 > 细胞分离和培养
作者: Xiurong Zhao
Xiurong ZhaoAffiliation: Stroke Program - Department of Neurology, The University of Texas Health Science Center at Houston, McGovern Medical School, Texas, USA
For correspondence: Xiurong.Zhao@uth.tmc.edu
Bio-protocol author page: a3091
Liyan Zhang
Liyan ZhangAffiliation: Stroke Program - Department of Neurology, The University of Texas Health Science Center at Houston, McGovern Medical School, Texas, USA
Bio-protocol author page: a3092
Shun-Ming Ting
Shun-Ming TingAffiliation: Stroke Program - Department of Neurology, The University of Texas Health Science Center at Houston, McGovern Medical School, Texas, USA
Bio-protocol author page: a3093
 and Jaroslaw Aronowski
Jaroslaw AronowskiAffiliation: Stroke Program - Department of Neurology, The University of Texas Health Science Center at Houston, McGovern Medical School, Texas, USA
Bio-protocol author page: a3094
Vol 6, Iss 8, 4/20/2016, 1421 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1795

[Abstract] Clearance of dead brain tissue including the dead neurons through phagocytosis is an endogenous function of microglia in the brain, which is critical for inflammation resolution after ischemic stroke or head trauma. By regulating the function or polarization status of microglia, we may control their phagocytosis efficacy and therefore the cleanup process for the dead brain tissue. We cultured rat cortical neurons and microglia from the same litter of embryos. The cultured neurons are subjected to irradiation for inducing neuronal apoptosis. After labeling with propidium iodide (PI), the dead neurons (DNs) are exposed to the cultured microglia for phagocytosis assay. By counting the number of DNs in each microglia, we calculate the phagocytosis index to quantify the phagocytosis efficacy of microglia toward DNs. The protocol is divided into 4 sections: A) culturing rat cortical neurons from pre-natal rat embryos, B) preparing dead neurons as phagocytosis target, C) culturing rat brain microglia, D) quantifying phagocytosis index of microglia toward the dead neurons.
Keywords: Microglia(小胶质细胞), Phagocytosis assay(吞噬试验), Apoptotic neurons(神经元凋亡)

[Abstract]

Materials and Reagents

  1. Cell Strainer (100 μm and 40 μm) (BD, Falcon, catalog number: 431752 and 431750 )
    Note: Currently, it is “Corning, Falcon, catalog number: 431752 and 431750”.
  2. Cell Lifter (VWR International, catalog number: 89030-910 )
  3. 100 mm Petri dish (Thermo Fisher Scientific, catalog number: 263991 )
  4. 60 mm TC dish (Corning, catalog number: CLS430166 )
  5. 75 cm2 TC flask (Corning, catalog number: 430825 )
  6. 24-well TC plate (BD, Falcon, catalog number: 353047 )
    Note: Currently, it is “Corning, Falcon, catalog number: 353047”.
  7. 1 ml Syringe (BD, Falcon, catalog number: 305217 )
  8. Glass Pasteur pipet (VWR International, catalog number: 14673-043 )
  9. E-18 Pregnant Sprague Dawley (SD) rat (Charles River Laboratories International)
  10. Pentobarbital (Sigma-Aldrich, catalog number: P-3761 )
  11. Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103 )
  12. B27 Supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17504 )
  13. Glutamine (Sigma-Aldrich, catalog number: G7513 )
  14. Penicillin/Streptomycin (100x) (GE Healthcare, Hyclone™, catalog number: SV30079 )
  15. DMEM (Corning, catalog number: 10-013-CM )
  16. Propidium Iodide (PI) (Sigma-Aldrich, catalog number: P-4170 )
  17. DAPI (4’, 6-Diamidino-2-Phenylindole, Dilactate) (Invitrogen, catalog number: D3571 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes™, catalog number: D3571”.
  18. 16% Paraformaldehyde (PFA) (Electron Microscopy Sciences, catalog number: 15170 )
  19. Fetal Bovine Serum (FBS) (GE Healthcare, Hyclone™, catalog number: SH30071 )
  20. Sodium pyruvate (Sigma-Aldrich, catalog number: P5280 )
  21. HEPES Buffer solution (Sigma-Aldrich, catalog number: 83264 )
  22. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
  23. Poly-D-Lysine (Sigma-Aldrich, catalog number: P0889 )
  24. HBSS (Lonza, catalog number: 10-543F )
  25. PBS (Corning, catalog number: 21-040-CV )
  26. 0.4% Trypan blue (Sigma-Aldrich, catalog number: T8154 )
  27. Alexa Fluor 488-Phalloidin (Invitrogen, catalog number: A12379 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes™, catalog number: A12379”.
  28. Mouse anti-MAP2 antibody (Sigma-Aldrich, catalog number: M4403 )
  29. Rabbit anti-mouse IgG-Alexa Fluor 488 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A11029 )
  30. Dissection buffer (see Recipes)
  31. DMEM/FBS (see Recipes)
  32. Neurobasal/B27 (see Recipes)
    Notes:
    1. All reagents and chemical for cell cultures must be sterile. Without special indication, all culture medium or buffer is used at room temperature (RT). And all operational procedures for neuronal culture and microglia (MΦ) culture, including animal dissecting, tissue triturating, cell plating or harvesting, and medium changing, have to be performed in a dissecting hood or cell culture hood using sterile tools.
    2. Poly-D-Lysine coated tissue culture (TC) plate. The TC dish and plate for the primary neuronal cells or purified microglia must be pre-coated with Poly-D-lysine (0.1 mg/ml in H2O) (2 ml for 60 mm2 TC dish, 0.5 ml per well for 24w-TC plate) for 30 min at 37 °C. After washing with sterile water, the TC dishes/plates have to be air-dried in the cell culture hood before use.
    3. The age of the embryos for cell culture. Although E16 embryos are more suitable for neuronal culture and post-natal 1-2 -day-old pups are more suitable for glia culture, we used the E-18 embryos to prepare the neuron culture and also the glial culture (to isolate MΦ). Thus, the neurons and MΦ are prepared from the same litter of embryos. The neuronal culture and microglia can be prepared from embryos or pups of different litters.
    4. The purity of neurons in the cortical neuronal culture. The Neurobasal/B27 medium is serum-free medium, which is optional for neuronal culture. However, there are other cell types in the cultures. In the 2-day-old neuronal cultures, the ratio of MAP2+-neurons is 43.6±23% (n=10, Figure 1).


      Figure 1. MAP2 immunofluorescence of 2-day-old rat cortical neurons in culture. The cortical neuron culture is fixed in 2% PFA and labeled by mouse anti-MAP2 antibody. The signals are visualized with Alexa Fluor 488 (Green). The nuclei of all cells are labeled with DAPI (red). The arrows indicate the MAP2+-neurons. Scale bar = 50 μm.

    5. All animal studies followed the guidelines outlined in Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and were approved by the Animal Welfare Committee of The University of Texas Health Science Center at Houston.
    6. The Irradiation operation followed the guidelines for Environment Health & Safety of The University of Texas Health Science Center at Houston.

Equipment

  1. Hemacytometer (VWR International, catalog number: 15170)
  2. Cell culture CO2 incubator (LabX, Sanyo, model: MCO-19AIC )
  3. Fluorescence microscopy with Imaging System, Olympus IX81 controlled by MetaMorph 7.4
  4. Dissecting microscopy (Nikon Corporation, model: SMZ-27 )
  5. Digital controlled orbital shaker (Thermo Fisher Scientific, model: MaxQTM 2000 )
  6. 137Cs Irradiation Source (J. L. SHEPHERD & ASSOCIATES, model: 143 ) (Figure 2)


    Figure 2. Photos of the 137Cs irradiator. The cultured neurons grown in the 60 mm TC dishes are loaded into the metal cylinder at top of the irradiator. Upon pressing the red button on the right controller, the cylinder will rotate and go down until completely being imbedded in the irradiator (the round bottom portion) for irradiation. By the irradiation time (the left controller) is over, the irradiator will automatically rotate the cylinder upward to its original position. By now, the irradiated cultures are ready to be removed from the cylinder and returned to the CO2 incubator.

Software

  1. MetaMorph software, version 7.4

Procedure

  1. Culturing rat cortical neurons
    1. Deeply anesthetize one E-18 pregnant rat by intraperitoneal injection of sodium pentobarbital (50 mg/kg).
    2. Prepare the surgical site with an appropriate skin disinfectant. We suggest 70% alcohol.
    3. Remove the embryos from uterus to one 100 mm Petri dish with 10 ml ice-cold Dissection Buffer.
    4. Remove the embryos’ brain using two surgical forceps and transfer the brains to another Petri dish with 10 ml ice-cold Dissection Buffer and leave the cells in dish on ice.
    5. Under a dissecting microscopy, after removing olfactory bulbs and meninges, the cortices are separated from other brain regions, and rinsed in ice-cold Neurobasal/B27 medium once. The dissected brain cortices may be divided into two portions: One portion will be used to culture neurons (see next step in this section) and another portion will be used to culture microglia (see Section C). 
    6. Transfer the cortices in a 100 μm cell strainer placed in a 100 mm petri dish containing 10 ml ice-cold Neurobasal/B27 medium and mince the tissue in the same medium by using a 1 ml-syringe plunger. 
    7. The dissociated cells that passing through the 100 μm cell strainer (which is placed in a 100 mm petri dish) are applied onto another 40 μm cell strainer, which is placed on top of a 50 ml centrifuge tube. 
    8. The collected cell suspension is subjected to a centrifugation step at 400 x g, 5 min. The pelleted cells are suspended in Neurobasal/B27 medium (from 5 rat embryos) are counted and seeded in Poly-D-lysine coated 60 mm TC dish (5 ml/dish) at a cell density of 2.5 x 107 cells/ml in Neurobasal/B27 medium (Kim et al., 2004). The yield of the cells is about 2.5 x 108 per brain, which can be seeded in 2 of 60 mm TC dishes.
    9. Culture the cells in a CO2 incubator (5% CO2, 21% O2) at 37.0 ± 0.5 °C for 2 d.
    10. After two days, the culture medium is completely replaced with fresh one and it is ready for irradiation treatment.

  2. Preparing dead neurons (DNs) as phagocytosis target
    1. The 2-day-old neuronal cells grown in 60 mm TC dishes from the above (step A10) are subjected to 137Cs irradiation, for 15 min (32Gy) (Kim et al., 2004). The culture dishes are automatically rotated during radiation to ensure uniform exposure. The irradiation operation is performed with supervision of the staff in the department of the Environment Health and Safety. 
    2. After irradiation, the cells are returned to the original CO2 incubator and cultured in the same culture medium for another 48 h. This incubation time after irradiation injury allows the injured cells progressively undergo apoptosis. Using Trypan Blue, MAP2 immunofluorescence and DAPI staining, we confirmed that ≥95% neurons at this stage are shrunken and dead (Figure 3). By comparing the cell number in the control dish (Figure 3A) and in the irradiation-treated dish (Figure 3B). There is no significantly neuronal loss. The immunofluorescence procedures for MAP2 followed the same method as we reported previously (Zhao et al., 2006).


      Figure 3. MAP2 immunofluorescence of 4-day-old rat cortical neurons in culture. A. Control; B. 2 d after irradiation injury. The cortical neuron cultures are fixed in 2% PFA and labeled by mouse anti-MAP2 antibody. The signals are visualized with Alexa Fluor 488 (green). The nuclei are highlighted with DAPI (red). Scale bar = 50 μm

    3. Label the dead neurons (DNs) in the culture dishes by adding 1 μg/ml of Propidium Iodide (PI) directly into the culture medium and incubating for 10 min in the CO2 incubator.
    4. Wash the cells with 10 ml PBS (37 °C), 3 times.
    5. After removing the PBS, add 3 ml Neurobasal/B27 medium (37 °C) into each dish and harvest the cells by using a Cell Lifter and then collect the cells in a 10 ml centrifuge tube.
    6. Triturate the cells using a Pasteur Pipet 5-10 strokes to ensure all cells to be dissociated.
    7. Apply the cell suspension through a 100 μm Cell Strainer and wash the strainer with 2 ml Neurobasal/B27 medium (37 °C).
    8. Collect all passed cells in one 15 ml centrifuge tube and centrifuge at 400 x g for 10 min.
    9. Re-suspend the cell pellet in 300 μl of DMEM/FBS per 60 mm TC dish and count the cell number using one hemocytometer. Then, adjust the cell density to 5 x 108 cells/ml in the same culture media. Now the PI-DNs are ready to be used as phagocytosis target at the next step for phagocytosis assay.
    10. Keep the PI-DNs at 4 °C fridge and wrap the tube with foil to protect the cells from light. The PI-DNs can be safely stored for one month.

  3. Culturing rat brain microglia (MΦ)
    Since we use embryos from the same litter to prepare the cortical neurons (Section A) and also the microglia (Section C), we usually distribute equal number of embryos to each culture. The beginning procedures for preparing microglia cultures are the same as the steps A1-7 described in Section A.
    1. This is next to the step A7 in Section A. The collected cell suspension is subjected to a centrifugation step at 400 x g, 5 min. The pelleted cells are suspended in DMEM/FBS (37 °C) at a ratio of 50 ml per brain and seeded in two 75 cm2 TC flasks (25 ml/flask).
    2. The glia cells are cultured in CO2 incubator (5% CO2, 21% O2) at 37.0 ± 0.5 °C. 
    3. The culture media are completely changed to fresh one on d 4, d 8 and d 12.
    4. On day 14, the astrocytes have grown into completely confluent. The MΦ become loosely attached or freely floating in the culture medium. We harvest MΦ by subjecting the flasks to a shaking procedure at 220 rpm for 10 min to detach MΦ from other glia cells.
    5. Collect the culture medium containing MΦ into 50 ml centrifuge tubes.
    6. Centrifuge at 400 x g, 10 min.
    7. After removing the supernatant, re-suspend the pelleted MΦ in 5 ml (per flask) fresh DMEM/FBS (37 °C).
    8. After cell counting, dilute the cells to 1 x 105 cells/ml with DMEM/FBS. The yield of the MΦ is about 1 - 2 x 106 cells per flask.
    9. Re-plate the MΦ in Poly-D-lysine coated 24w-TC plate at 5 x 104 cells in 500 μl/well.
    10. Culture the purified MΦ in CO2 incubator (5% CO2, 21% O2) at 37.0 ± 0.5 °C for 24 h. And by now it is ready to be used at the next step for phagocytosis assay. By immunofluorescence, we confirmed that more than 95% of the cells are CD68+-MΦ and CD11b+- MΦ (Zhao et al., 2007).

  4. Quantifying phagocytosis efficacy
    1. Adding 10 μl of the PI-DNs (prepared at step B10) into the re-plated MΦ cultures (prepared at step C10) at a target/effector ratio of 100:1 (DNs: MΦ).
    2. Incubate the cells in CO2 incubator (5% CO2, 21% O2) at 37.0 ± 0.5 °C for 2 h.
    3. At end of incubation, remove the free-floating PI-DNs by aspirating the culture medium and fix the cells with 4% room temperature paraformaldehyde (PFA) for 10 min. It is not necessary to have a washing step prior to fixture.
    4. Rinse the cells by adding 1 ml PBS in each well, 3 times.
    5. Incubate the cells in 1 ml of 0.1% Triton X-100 in PBS for 1 min.
    6. Rinse the cells with 1 ml PBS per well, 3 times.
    7. Label the cells with 300 μl of Alexa Fluor 488-Phalloidin at 1:100 dilution and 1 μg/ml DAPI in PBS for 30 min.
    8. Rinse the cells with 1 ml PBS, 3 times.
    9. Observe the cells under a fluorescence microscopy and acquire the images using the filter sets at Ex/Em of 490/520 nm for Phalloidin-labeled MΦ, Ex/Em of 550/575 nm for PI-DNs and Ex/Em of 365/480 nm for DAPI-labeled nuclei.
    10. By combining the images acquired with the above 3 sets of filters at the same position, we may clearly observe the PI-DNs within each MΦ. The phagocytized DNs are fluorescently red within Phalloidin-labeled green MΦ. The nuclei of the MΦ are blue (Figure 4). Five images under 40x objective are recorded. The number of the PI-positive neuronal nuclei in each MΦ is counted manually, on still images. At least fifty MΦ cells from each condition should be analyzed. The average number of phagocytized dead neurons (PI-DNs) within each MΦ is calculated and used as Phagocytosis Index (Zhao et al., 2015).


    Figure 4. Phalloidin-labeled microglia (green) after phagocytizing the PI-DNs (red). The nucleus is labeled with DAPI (blue). Scale bar = 20 μm. The arrows indicate the phagocytized PI-DNs within the MΦ.

Notes

  1. The incubation time of MΦ with dead neurons. We subject MΦ to PI-DNs for 2 h to measure the Phagocytosis Index. To precisely quantify the phagocytosis efficacy, it is necessary to perform a time course (1-4 h) study.
  2. The number of cell counting. The size of DNs is not unique and the number within each MΦ varies. Therefore, counting the number of DNs in each MΦ should measure all MΦ cells within the image field. We count 50 of MΦ for each experimental condition. However, more number counted, more precise data.
  3. Bias of image acquisition. To determine the phagocytosis capacity of the MΦ toward the PI-DNs, we suggest using a microscopy with a motorized stage and CCD camera, which is controlled by a computer software (e.g., MetaMorph software) that may allow image stitching. This computer-controlled image acquisition system may permit automatic and unbiased cell counting. However, the automatically counted data needs to be confirmed manually. And when multiple batches of cell counting are repeated, it is better done by the same person.

Recipes

  1. Dissection buffer
    HBSS
    4.2 mM bicarbonate
    1 mM pyruvate
    20 mM HEPES
    1 mg/ml BSA
    1x Penicillin/Streptomycin (pH 7.25)
  2. DMEM/FBS
    DMEM
    10% FBS
    1x Penicillin/Streptomycin
  3. Neurobasal/B27
    Neurobasal culture medium
    1x B27 supplement
    0.5 mM L-Glutamine
    1x Penicillin/Streptomycin

Acknowledgments

This protocol is supported in part by grants NS060768, NS064109 and NS084292 of National Institute of Health, National Institute of Neurological Disorders and Stroke. For citation, please refer to Zhao et al. (2015).

References

  1. Kim, D. H., Zhao, X., Tu, C. H., Casaccia-Bonnefil, P. and Chao, M. V. (2004). Prevention of apoptotic but not necrotic cell death following neuronal injury by neurotrophins signaling through the tyrosine kinase receptor. J Neurosurg 100(1): 79-87.
  2. Zhao, X., Ou, Z., Grotta, J. C., Waxham, N. and Aronowski, J. (2006). Peroxisome-proliferator-activated receptor-gamma (PPARgamma) activation protects neurons from NMDA excitotoxicity. Brain Res 1073-1074: 460-469.
  3. Zhao, X., Sun, G., Zhang, J., Strong, R., Song, W., Gonzales, N., Grotta, J. C. and Aronowski, J. (2007). Hematoma resolution as a target for intracerebral hemorrhage treatment: role for peroxisome proliferator-activated receptor gamma in microglia/macrophages. Ann Neurol 61(4): 352-362.
  4. Zhao, X., Wang, H., Sun, G., Zhang, J., Edwards, N. J. and Aronowski, J. (2015). Neuronal interleukin-4 as a modulator of microglial pathways and ischemic brain damage. J Neurosci 35(32): 11281-11291.

材料和试剂

  1. 细胞过滤器(100μm和40μm)(BD,Falcon,目录号:431752和431750)
    注意:目前为"Corning,Falcon,目录号:431752和431750"。
  2. 电池升降器(VWR International,目录号:89030-910)
  3. 100mm培养皿(Thermo Fisher Scientific,目录号:263991)
  4. 60mm TC皿(Corning,目录号:CLS430166)
  5. 75cm 2 TC烧瓶(Corning,目录号:430825)
  6. 24孔TC板(BD,Falcon,目录号:353047) 注意:目前为"Corning,Falcon,目录号:353047"。
  7. 1ml注射器(BD,Falcon,目录号:305217)
  8. 玻璃巴斯德吸管(VWR International,目录号:14673-043)
  9. E-18怀孕Sprague Dawley(SD)大鼠(Charles River Laboratories International)
  10. 戊巴比妥(Sigma-Aldrich,目录号:P-3761)
  11. Neurobasal培养基(Thermo Fisher Scientific,GibcoTM,目录号:21103)
  12. B27补充(Thermo Fisher Scientific,GibcoTM,目录号:17504)
  13. 谷氨酰胺(Sigma-Aldrich,目录号:G7513)
  14. 青霉素/链霉素(100x)(GE Healthcare,Hyclone TM,目录号:SV30079)
  15. DMEM(Corning,目录号:10-013-CM)
  16. 碘化丙啶(PI)(Sigma-Aldrich,目录号:P-4170)
  17. DAPI(4',6-二脒基-2-苯基吲哚,二乳酸盐)(Invitrogen,目录号:D3571)
    注意:目前,它是"Thermo Fisher Scientific,Molecular Probes?,目录号:D3571"。
  18. 16%多聚甲醛(PFA)(Electron Microscopy Sciences,目录号:15170)
  19. 胎牛血清(FBS)(GE Healthcare,Hyclone TM,目录号:SH30071)
  20. 丙酮酸钠(Sigma-Aldrich,目录号:P5280)
  21. HEPES缓冲液(Sigma-Aldrich,目录号:83264)
  22. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
  23. 聚-D-赖氨酸(Sigma-Aldrich,目录号:P0889)
  24. HBSS(Lonza,目录号:10-543F)
  25. PBS(Corning,目录号:21-040-CV)
  26. 0.4%台盼蓝(Sigma-Aldrich,目录号:T8154)
  27. Alexa Fluor 488-Phalloidin(Invitrogen,目录号:A12379) 注意:目前,它是"Thermo Fisher Scientific,Molecular Probes?,目录号:A12379"。
  28. 小鼠抗MAP2抗体(Sigma-Aldrich,目录号:M4403)
  29. 兔抗小鼠IgG-Alexa Fluor 488(Thermo Fisher Scientific,InvitrogenTM,目录号:A11029)
  30. 解剖缓冲区(参见配方)
  31. DMEM/FBS(参见配方)
  32. Neurobasal/B27(见配方)
    注意:
    1. 用于细胞培养的所有试剂和化学品必须是无菌的。没有 特殊适应症,所有培养基或缓冲液在室内使用 温度(RT)。和所有操作程序的神经元文化 和小胶质细胞(MΦ)培养,包括动物解剖,组织 研磨,细胞电镀或收获,以及培养基更换 ?在解剖罩或细胞培养罩中使用无菌进行 工具。
    2. 聚-D-赖氨酸包被的组织培养(TC)板。 TC盘和板材 对于原代神经元细胞或纯化的小胶质细胞必须预包被 与聚-D-赖氨酸(在H 2 O中为0.1mg/ml)(对于60mm皿2ml,2ml),0.5ml 每孔24w-TC板)在37℃下30分钟。洗涤后 无菌水,TC培养皿/板必须在细胞中风干 文化罩。
    3. 胚胎细胞培养的年龄。虽然E16胚胎更多 适合神经元培养和产后1-2天龄的幼仔更多 适合胶质细胞培养,我们用E-18胚胎做准备 神经元培养和神经胶质培养(以分离MΦ)。因此, 神经元和MΦ从同样的胚胎胚胎制备。的 神经元培养和小胶质细胞可以从胚胎或幼崽制备 不同窝。
    4. 皮质神经元培养中神经元的纯度。的 Neurobasal/B27培养基是无血清培养基,其是任选的 神经元文化。然而,在培养物中存在其他细胞类型。 在2天龄的神经元培养物中,MAP 2 +神经元的比率为43.6±23% (n = 10,图1)。


      图1. 2日龄大鼠皮质神经元的MAP2免疫荧光 培养。皮层神经元培养物在2%PFA中固定,并标记 小鼠抗MAP2抗体。信号用Alexa Fluor显现 488(绿色)。所有细胞的核用DAPI(红色)标记。的 箭头表示MAP 2 + - 神经元。比例尺=50μm。

    5. 所有动物研究都遵循指南中所述的指南 护理和使用的国家研究所的实验动物 健康和批准的动物福利委员会 德克萨斯大学健康科学中心在休斯敦。
    6. 辐射操作遵循环境健康指南 ?&德克萨斯大学健康科学中心的安全性 休斯顿。

设备

  1. 血细胞计数器(VWR International,目录号:15170)
  2. 细胞培养CO 2培养箱(LabX,Sanyo,型号:MCO-19AIC)
  3. 荧光显微镜与成像系统,奥林巴斯IX81由MetaMorph 7.4控制
  4. 解剖显微镜(尼康公司,型号:SMZ-27)
  5. 数字控制轨道摇床(Thermo Fisher Scientific,型号:MaxQ TM 2000)
  6. < sup> 137 Cs照射源(J.L.SHEPHERD& ASSOCIATES,型号:143)(图2)

    图2. 137 Cs辐射器的照片。在60 mm TC培养皿中培养的神经元加载进入辐射器顶部的金属圆筒中。在按下右侧控制器上的红色按钮时,气缸将旋转并下降,直到完全嵌入辐照器(圆底部)中以进行照射。通过照射时间(左控制器)结束,辐照器将自动将圆筒向上旋转到其原始位置。到现在,辐照的培养物准备从圆筒中取出并返回CO 2培养箱。

软件

  1. MetaMorph软件,版本7.4

程序

  1. 培养大鼠皮层神经元
    1. 通过腹膜内注射戊巴比妥钠(50mg/kg),对一只E-18怀孕大鼠进行深度麻醉。
    2. 使用适当的皮??肤消毒剂准备手术部位。我们建议70%的酒精。
    3. 删除胚胎从子宫到一个100毫米培养皿与10毫升冰冷的解剖缓冲液。
    4. 删除胚胎的大脑使用两个手术镊子,并将大脑转移到另一个培养皿与10毫升冰冷的解剖缓冲液,并将细胞在冰上的菜。
    5. 在解剖显微镜下,在除去嗅球和脑膜后,将皮质与其他脑区域分离,并在冰冷的Neurobasal/B27培养基中漂洗一次。解剖的脑皮质可以分为两部分:一部分将用于培养神经元(参见本节中的下一步),另一部分将用于培养小胶质细胞(参见C节)。
    6. 将皮层转移到放置在含有10ml冰冷的Neurobasal/B27培养基的100mm培养皿中的100μm细胞过滤器中,并通过使用1ml注射器柱塞在相同培养基中切碎组织。
    7. 将通过100μm细胞过滤器(其放置在100mm培养皿中)的离解细胞施加到另一个40μm细胞过滤器上,所述细胞过滤器置于50ml离心管的顶部。
    8. 将收集的细胞悬浮液在400×g下进行离心步骤5分钟。将沉淀的细胞悬浮于Neurobasal/B27培养基(来自5个大鼠胚胎)中,并以2.5×10 7个细胞密度接种在聚-D-赖氨酸包被的60mm TC培养皿(5ml /皿)/NE27细胞/ml,在Neurobasal/B27培养基中(Kim等人,2004)。细胞的产量为每个大约2.5×10 8个/sup,其可以接种在2个60mm TC培养皿中。
    9. 在37.0±0.5℃下在CO 2培养箱(5%CO 2,21%O 2)中培养细胞2天。
    10. 两天后,将培养基完全更换为新鲜培养基,并准备进行辐照处理。

  2. 准备死亡神经元(DNs)作为吞噬靶点
    1. 将来自上述(步骤A10)的60mm TC培养皿中生长的2日龄神经元细胞进行15分钟(32Gy)的 Cs照射15分钟(Kim等,/em,2004)。培养皿在辐射期间自动旋转以确保均匀暴露。辐射操作是在环境健康和安全部门的工作人员的监督下进行的。
    2. 照射后,将细胞返回到原始CO 2培养箱中,并在相同的培养基中再培养48小时。照射损伤后的这种孵育时间允许损伤的细胞逐渐经历细胞凋亡。使用台盼蓝,MAP2免疫荧光和DAPI染色,我们证实在这一阶段的≥95%的神经元收缩和死亡(图3)。通过比较对照组(图3A)和照射处理组(图3B)中的细胞数。没有显着的神经元损失。 MAP2的免疫荧光程序遵循与我们先前报道的相同的方法(Zhao等人,2006)。


      图3. 4天龄大鼠皮质神经元的MAP2免疫荧光 文化。 A。控制; B.放射损伤后2 d。皮层 神经元培养物在2%PFA中固定并用小鼠抗MAP2标记 抗体。用Alexa Fluor 488(绿色)显示信号。的 核用DAPI(红色)突出显示。比例尺=50μm

    3. 通过将1μg/ml碘化丙啶(PI)直接添加到培养基中并在CO 2培养箱中孵育10分钟来标记培养皿中的死亡神经元(DN)。
    4. 用10ml PBS(37℃)洗涤细胞,3次。
    5. 除去PBS后,加入3毫升Neurobasal/B27培养基(37℃)到每个菜,收获细胞通过使用细胞升降器,然后收集在10ml离心管中的细胞。
    6. 使用巴斯德吸管5-10次敲击细胞以确保所有细胞被解离。
    7. 应用细胞悬浮液通过100μm细胞过滤器和用2ml Neurobasal/B27培养基(37℃)洗涤过滤器。
    8. 收集所有通过的细胞在一个15毫升离心管和400×g离心10分钟。
    9. 重悬细胞沉淀在300微升的DMEM/FBS每60毫米TC皿和计数细胞数量使用一个血细胞计数器。然后,在相同的培养基中将细胞密度调节至5×10 8个细胞/ml。现在,PI-DNs准备好在吞噬作用测定的下一步骤用作吞噬作用靶。
    10. 保持PI-DNs在4°C冰箱和用箔包裹管,以保护细胞免受光。 PI-DN可以安全地存储一个月。

  3. 培养大鼠脑小胶质细胞(MΦ)
    由于我们使用来自相同垃圾的胚胎来制备皮层神经元(部分A)和小胶质细胞(部分C),我们通常将相等数量的胚分配给每个培养物。制备小胶质细胞培养物的起始程序与A部分中描述的步骤A1-7相同。
    1. 这是在部分A中的步骤A7旁边。将收集的细胞悬浮液进行400×g离心5分钟的离心步骤。将沉淀的细胞以每个脑50ml的比例悬浮在DMEM/FBS(37℃)中,并接种在两个75cm 2 TC烧瓶(25ml /烧瓶)中。
    2. 将神经胶质细胞在37.0±0.5℃下在CO 2培养箱(5%CO 2,21%O 2)中培养。
    3. 培养基在d 4,d 8和d 12时完全改变为新鲜培养基。
    4. 在第14天,星形胶质细胞生长成完全汇合。 MΦ松散地附着或自由漂浮在培养基中。我们通过使烧瓶经受以220rpm的摇动程序10分钟以从其它神经胶质细胞分离MΦ而收获MΦ。
    5. 收集含有MΦ的培养基到50ml离心管中。
    6. 以400×g离心,10分钟。
    7. 除去上清液后,将沉淀的MΦ重新悬浮于5ml(每瓶)新鲜DMEM/FBS(37℃)中。
    8. 细胞计数后,用DMEM/FBS将细胞稀释至1×10 5个细胞/ml。 MΦ的产量为每个烧瓶约1-2×10 6个细胞。
    9. 在500μl/孔中以5×10 4个细胞重新铺板聚-D-赖氨酸包被的24w-TC板中的MΦ。
    10. 将纯化的MΦ在37℃±0.5℃下在CO 2培养箱(5%CO 2,21%O 2)中培养24小时。并且现在它准备好在下一步用于吞噬测定。通过免疫荧光,我们证实超过95%的细胞是CD68高 - 和 - CD11b - 高 - - - MΦ(Zhao等人, 2007)。

  4. 定量吞噬功效
    1. 将10μlPI-DN(在步骤B10制备)以100:1的目标/效应物比例(DNs:MΦ)加入重新镀覆的MΦ培养物(在步骤C10制备)。
    2. 将细胞在37℃±0.5℃下在CO 2培养箱(5%CO 2,21%O 2)中孵育2小时。
    3. 在孵育结束时,通过吸出培养基和用4%室温多聚甲醛(PFA)固定细胞10分钟,去除自由浮动PI-DNs。在固定之前没有必要具有洗涤步骤。
    4. 通过在每个孔中加入1ml PBS冲洗细胞,3次。
    5. 孵育细胞在1ml的0.1%Triton X-100在PBS中1分钟。
    6. 用1ml PBS每孔冲洗细胞,3次。
    7. 用300μlAlexa Fluor 488-Phalloidin(1:100稀释)和1μg/ml DAPI(在PBS中)标记细胞30分钟。
    8. 用1ml PBS冲洗细胞,3次。
    9. 在荧光显微镜下观察细胞,并使用在490/520nm的Ex/Em的鬼影肽标记的MΦ的Ex/Em,对于PI-DN的550/575nm的Ex/Em和对于365/480的Ex/Em, nm为DAPI标记的核。
    10. 通过将在上述3组滤波器获得的图像组合在相同位置,我们可以清楚地观察每个MΦ内的PI-DN。吞噬的DN在鬼笔环肽标记的绿色MΦ内是荧光红色的。 MΦ的核是蓝色的(图4)。记录40x物镜下的五个图像。在静止图像上手动计数每个MΦ中的PI阳性神经元核的数目。应当分析来自每个条件的至少50个MΦ细胞。计算每个MΦ内吞噬的死亡神经元(PI-DN)的平均数,并用作吞噬作用指数(Zhao et al。,2015)。


    图4.吞噬PI-DN(红色)后的鬼笔环肽标记的小胶质细胞(绿色)。细胞核用DAPI(蓝色)标记。比例尺=20μm。箭头表示在MΦ内的吞噬的PI-DN

笔记

  1. MΦ与死亡神经元的孵育时间。我们对MΦ进行PI-DNs 2小时,以测量吞噬指数。为了精确地量化吞噬功效,有必要进行时间过程(1-4小时)研究。
  2. 细胞计数的数量。 DN的大小不是唯一的,并且每个MΦ内的数量变化。因此,计算每个MΦ中的DN数量应该测量图像场内的所有MΦ单元。对于每个实验条件,我们计数MΦ。但是,更多数量,更精确的数据。
  3. 图像采集的偏差。为了确定MΦ对PI-DN的吞噬能力,??我们建议使用具有机动载物台和CCD照相机的显微镜,其由计算机软件(例如,MetaMorph软件)控制,其可允许图像拼接。该计算机控制的图像采集系统可以允许自动和不偏置的细胞计数。但是,需要手动确认自动计数的数据。当重复多批次的细胞计数时,最好由同一个人完成。

食谱

  1. 解剖缓冲器
    HBSS
    4.2 mM碳酸氢盐 1mM丙酮酸 20 mM HEPES
    1mg/ml BSA
    1×青霉素/链霉素(pH 7.25)
  2. DMEM/FBS
    DMEM
    10%FBS
    1×青霉素/链霉素
  3. Neurobasal/B27
    神经基础培养基
    1x B27补充
    0.5mM L-谷氨酰胺 1×青霉素/链霉素

致谢

该协议部分地由National Institute of Health,National Institute of Neurological Disorders and Stroke的授权NS060768,NS064109和NS084292支持。有关引用,请参阅Zhao 等(2015)。

参考文献

  1. Kim,D.H.,Zhao,X.,Tu,C.H.,Casaccia-Bonnefil,P.and Chao,M.V。(2004)。 预防神经营养因子通过酪氨酸激酶受体信号传导的神经元损伤后的凋亡而非坏死性细胞死亡。/a> J Neurosurg 100(1):79-87
  2. Zhao,X.,Ou,Z.,Grotta,J.C.,Waxham,N。和Aronowski,J。(2006)。 Peroxisome-proliferator-activated receptor-gamma(PPARgamma)activation保护神经元免受NMDA兴奋性毒性。 Brain Res 1073-1074:460-469。
  3. Zhao,X.,Sun,G.,Zhang,J.,Strong,R.,Song,W.,Gonzales,N.,Grotta,J.C.and Aronowski,J。(2007)。 血肿分辨率作为脑内出血治疗的目标:过氧化物酶体增殖物激活受体γ在小胶质细胞中的作用/巨噬细胞。 Ann Neurol 61(4):352-362
  4. Zhao,X.,Wang,H.,Sun,G.,Zhang,J.,Edwards,N.J.and Aronowski,J.(2015)。 神经元白细胞介素-4作为小胶质细胞通路和缺血性脑损伤的调节剂 J Neurosci 35(32):11281-11291。
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How to cite this protocol: Zhao, X., Zhang, L., Ting, S. and Aronowski, J. (2016). Phagocytosis Assay of Microglia for Dead Neurons in Primary Rat Brain Cell Cultures. Bio-protocol 6(8): e1795. DOI: 10.21769/BioProtoc.1795; Full Text



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