搜索

An in vitro Model of Neuron-macrophage Interaction to Generate Macrophages with Neurite Outgrowth Properties
使用神经元-巨噬细胞相互作用体外模型生成具有神经突起生长特性的巨噬细胞   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Macrophages are known to play beneficial roles in axon regeneration after nerve injury. To develop an in vitro model in which injury signals can elicit pro-regenerative macrophage activation, we established co-cultures consisting of adult dorsal root ganglia sensory neurons and peritoneal macrophages and added cAMP analogue dibutyryl cAMP. The conditioned medium collected from the co-cultures exhibited robust neurite outgrowth activities. The neurite outgrowth activities were almost completely abrogated by addition of minocycline, a macrophage deactivator, indicating that factors responsible for neurite outgrowth are produced by activated macrophages.

Background

CNS neurons of adult mammals do not spontaneously regenerate axons after injury. Preconditioning peripheral nerve injury allows the dorsal root ganglia (DRG) sensory axons to regenerate central branches by promoting expression of regeneration-associated genes. We have previously showed that activated macrophages in the DRG following preconditioning injury critically contribute to enhancement intrinsic regeneration capacity of the DRG sensory neuron (Kwon et al., 2013). To identify molecular factors involved in the activation of macrophages following nerve injury, we have developed an in vitro model in which neuron-macrophages interactions are elicited by cAMP, a well-known reagent to enhance regenerative capacity of neurons. Compared to the previous model to activate macrophages using zymosan, our model utilizes a more physiologic stimulus resembling molecular events in the preconditioning peripheral injury model.

Materials and Reagents

  1. 1.5 ml Eppendorf tubes
  2. 15 ml conical tube
  3. Cell culture insert with 0.4 μm transparent PET membrane (Corning, Falcon®, catalog number: 353090 )
  4. 70 μm nylon cell strainer (Corning, Falcon®, catalog number: 352350 )
  5. 50 ml conical tube
  6. 6 well plate
  7. 0.2 μm filter (BD)
  8. 8-well culture slide (Corning, BiocoatTM, catalog number: 354632 )
  9. Dulbecco’s modified Eagle medium (DMEM) (GE Healthcare, HycloneTM, catalog number: SH30243.01 )
  10. Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C9407-100MG )
  11. Neurobasal® medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103-049 )
  12. B-27® supplement (50x), serum free (Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044 )
  13. GlutaMaxTM (Thermo Fisher Scientific, GibcoTM, catalog number: 35050-061 )
  14. Penicillin-streptomycin (10,000 U/ml, 10,000 µg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  15. Poly-D-lysine hydrobromide (Sigma-Aldrich, catalog number: P6407-5MG )
  16. Laminin (Thermo Fisher Scientific, GibcoTM, catalog number: 23017-015 )
  17. Phosphate-buffered saline (PBS) (GE Healthcare, HycloneTM, catalog number: SH30256.01 )
  18. Red blood cell lysis buffer (QIAGEN, catalog number: 158904 )
  19. 10% FBS (GE Healthcare, HycloneTM, catalog number: SH30919.03 )
  20. Adenosine 3’,5’-cyclic monophosphate, N6,O2’-dibutyryl-, sodium salt (db-cAMP; 100 μM) (EMD Millipore, Calbiochem®, catalog number: 28745 )
  21. Minocycline (Sigma-Aldrich, catalog number: M9511 )
  22. Paraformaldehyde-13C (Sigma-Aldrich, catalog number: 604380 )
  23. Normal goat serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16210072 )
  24. Triton X-100 (DAEJUNG CHEMICAL & MITALS, catalog number: 8566-4405 )
  25. Goat anti-mouse IgG (H + L) secondary antibody, Alexa Fluor® 594 conjugate (Thermo Fisher Scientific, InvitrogenTM, catalog number: A11005 )
  26. Anti β III tubulin (Tuj-1) (Promega, catalog number: G7121 )
  27. Neuron culture medium (see Recipes)
  28. Macrophage culture medium (see Recipes)

Equipment

  1. Fine scissors
  2. Fine forceps
  3. Hemacytometer (Marienfeld-Superior)
  4. Dissecting stereomicroscope (Carl Zeiss, model: Stemi DV4 )
  5. Cell culture CO2 incubator (Panasonic)
  6. Twist shaker (FINEPCR, model: Twist shaker Tw3t )
  7. Tabletop centrifuge (Sorvall)
  8. Pipette-aid
  9. Confocal microscope (Olympus, model: IX71 )

Software

  1. ImageJ (http://rsbweb.nih.gov/ij/index.html)

Procedure

  1. Adult DRG neurons are obtained from adult C57BL6 mice. After animals are sacrificed, skin and paravertebral muscles are dissected to expose the vertebral column. Bilateral dorsal root ganglia (DRGs) from the S1 up to the C1 level are freshly dissected using fine scissors and forceps under a dissecting microscope and soaked into fresh cold DMEM. Make sure to be careful not to damage DRGs when detaching the DRGs from the roots and nerves.
    Note: It is critically important to dissect DRGs as quickly as possible. The collection of the total DRGs from one mouse should not take longer than 30 min.
  2. Transfer DRGs to a 1.5 ml Eppendorf tube with a blue pipette tip with an end cut off. Quickly spin down DRGs and aspirate off DMEM. Add 1 ml of DMEM containing 125 U/ml type XI collagenase and incubate for 90 min at 37 °C with gentle rotation using a twist shaker kept in an incubator. The optimal range for rotation speed is around 35-45 rpm.
  3. Aspirate off collagenase-containing DMEM and resuspend DRGs with 1 ml of DMEM. When DMEM is added, DRGs will float. Wait until DRGs fall down, and then aspirate off. Repeat this step at least five times to remove collagenase completely.
    Note: Try not to aspirate off completely. It is important to prevent DRGs from being sucked out.
  4. Transfer DRGs to 15 ml conical tube using a cut off blue pipette tip. Then triturate at least 15 times using a blue tip until a homogenous suspension of cells is achieved.
    Note: Trituration should be very gentle. Avoid making bubbles and try not to touch the bottom of conical tube.
  5. Centrifuge the tube at about 239 x g for 3 min to remove cell debris in supernatant. Resuspend cell pellets in 1 ml Neurobasal medium supplemented with B27 and triturate 5 to 10 times. Then pass the cells through a 70 μm cell strainer installed on a 50 ml conical tube. Wait for 2 min, and then pour Neurobasal/B-27 medium using a pipette-aid to the strainer so that cells passing the strainer come off easily. Wait one more minute.
  6. Plate dissociated cells onto a 6 well plate pre-coated with 0.01% poly-D-lysine and laminin (3 μg/ml), and shake the plate very gently for the cells to be evenly distributed. A total of 6 x 106 DRG neurons are plated for each well. Then, the plate is placed in a CO2 incubator maintained at 37 °C.
    Note: Poly-D-lysine (0.01%) and laminin (3 μg/ml) are diluted with distilled water. Poly-D-lysine coats the plate for 2 h at 37 °C or overnight at 4 °C, after which the plate is washed twice with distilled water. Then, the plate is coated with laminin for 2 h at room temperature, and washed twice with PBS. Then the plate is dried at room temperature before cell plating.
  7. The co-cultures need to be set up 4 h after the initial plating of dissociated DRG neurons. Primary peritoneal macrophages are prepared from adult C57BL6 mice. After sacrificing mice in CO2 chamber, abdominal skin is delicately dissected to expose the peritoneum.
  8. Inject 10 ml of ice-cold PBS into the peritoneum. Gently massage the peritoneum for 1-2 min. Then, suck out PBS and transfer to a new 50 ml conical tube.
  9. The lavage fluid is centrifuged at 239 x g for 10 min at 4 °C to pellet the cells. The cell pellets are resuspended in 3 ml of the red blood cell lysis buffer for 3 min at room temperature (RT) and then centrifuged at 239 x g for 10 min at 4 °C to pellet the cells again.
  10. Pelleted cells are resuspended in macrophage culture medium. The peritoneal macrophages are plated on a cell culture insert placed on the 6 well plate where dissociated DRG neurons have been plated 4 h before. The number of plated macrophages is kept 5 times higher than that of cultured DRG neurons.
  11. 4 h after macrophage plating, the neuron–macrophage co-cultures are treated with dibutyryl-cAMP (db-cAMP; 100 μM) or PBS as a control. In some wells, macrophage deactivator minocycline is added with db-cAMP at a concentration of 10 μg/ml.
  12. After 24 h, the culture medium is replaced with fresh macrophage culture medium. The co-cultures are maintained for 72 h without changing the medium.
  13. Then the conditioned medium is collected, centrifuged at 239 x g for 5 min, and passed through a 0.2 μm filter to remove any remaining cellular debris. The collected conditioned medium is stored at -70 °C until use.
  14. For neurite outgrowth assays, adult DRG neurons are obtained and dissociated using the same method described in steps 1-3.
  15. Cell pellets are resuspended in Neurobasal-A supplemented with B27 and a total of 1 x 104 cells (per well) are plated onto eight-well culture slides pre-coated with 0.01% poly-D-lysine and 3 μg/ml laminin. Precoating is done using the same method described in step 6.
  16. The neuron cultures are maintained in an incubator for 2 h allowing for cell attachment to the bottom. And the culture medium is replaced with the thawed conditioned medium collected in step 15.
  17. After 15 h from the initial plating, the cells are directly fixed with 4% cold paraformaldehyde for 20 min.
  18. The fixed cells on slides are washed three times with 1x PBS and incubated with a blocking solution consisting of 10% NGS and 0.1% Triton-X for 30 min. Then, wash with 1x PBS twice, perform primary antibody (Tuj-1) staining at 1:1,000 dilution with 10% NGS for 4 h at RT or overnight at 4 °C.
  19. Wash twice with 1x PBS, and perform secondary antibody (goat-anti rabbit) staining at 1:500 dilution with 10% NGS for 2 h at RT. Then, wash the slides twice with 1x PBS and mount the coverslips on the slides with mounting gel.
  20. Take images using confocal microscope for immunostained β III tubulin to visualize neurite outgrowth.

Data analysis

  1. The mean neurite length per neuron is measured to compare the extent of neurite outgrowth induced by different conditioned medium. Neurite length is measured using the Neuron J plugin for the image analysis software suite NIH ImageJ (publicly available from http://rsbweb.nih.gov/ij/index.html), which facilitates tracing and quantification of elongated structures. Each experimental condition is replicated in two wells per culture. Each well is divided into four quadrants, and a 200x magnification image is obtained at the center of each quadrant (four images in each well). Neurites are traced for all neurons in each image, and the number of DRG neurons per image is counted manually. Approximately 20 neurons per image are observed, and, therefore, around 80 neurons are measured for each well. The average neurite length per neuron in each well is calculated by dividing the total neurite length from the total number of neurons.
  2. Conditioned medium collected from the neuron-macrophage co-cultures treated with PBS do not support neurite outgrowth (Figure 1A). When DRG neurons are grown with conditioned medium treated with dibutyryl cAMP, they invariable show robust neurite outgrowth (Figure 1B). If minocycline, a macrophage deactivator, is added to the neuron-macrophage co-cultures along with cAMP, neurite outgrowth activity is almost completely abolished, suggesting that the neurite outgrowth activity required activation of macrophage (Figure 1C). In our previous study, we showed that conditioned medium collected from either neuron or macrophage alone cultures with cAMP treatment did not support neurite outgrowth (Kwon et al., 2013). Therefore, the interaction between neurons and macrophages are critical for the neurite outgrowth activity.


    Figure 1. Neuron-macrophage interactions promote neurite outgrowth activity. Neuron (adult DRG neurons) and peritoneal macrophage co-cultures were treated with PBS (A), cAMP (B) and cAMP + minocycline (C). Scale bars = 100 μm.

Notes

  1. In the neurite outgrowth assay using this protocol, DRG neurons do not grow any significant neurites within 15 h in culture. However, if DRG neurons are allowed to grow longer than 15 h, some degree of neurite outgrowth can be observed even in control condition. Similar findings were reported in the neurite outgrowth assay using rat DRG neurons in the previous study (Cafferty et al., 2004). In pilot experiments, we test several different culture durations, and we found that 15 h of culture resulted in minimal neurite outgrowth in control condition while highly robust neurite outgrowth was achieved with conditioned medium treated with cAMP.
  2. To set up the neuron-macrophage co-cultures, dissociated macrophages can be added directly to cultured DRG neurons without cell culture insert. In our previous study, we directly compared the extent of neurite outgrowth between conditioned medium collected from direct co-cultures and from the co-cultures with the two cell types separated by cell culture insert (Kwon et al., 2013), and there was no difference between the two conditions. This suggests that the two cell types are communicating with each other using soluble molecules. The following study in our lab discovered that CCL2, secreted from neurons, is responsible to activate macrophages into a pro-regenerative phenotype in this co-culture model (Kwon et al., 2015).

Recipes

  1. Neuron culture medium
    Neurobasal medium (500 ml)
    2% B27
    1% glutamax
    1% penicillin-streptomycin
  2. Macrophage culture medium
    DMEM
    10% FBS
    1% penicillin-streptomycin

Acknowledgments

This protocol is supported by a grant NRF-2015R1A2A1A01003410 from the Ministry of Science, ICT and Future Planning, Republic of Korea.

References

  1. Cafferty, W. B., Gardiner, N. J., Das, P., Qiu, J., McMahon, S. B. and Thompson, S. W. (2004). Conditioning injury-induced spinal axon regeneration fails in interleukin-6 knock-out mice. J Neurosci 24(18): 4432-4443.
  2. Kwon, M. J., Kim, J., Shin, H., Jeong, S. R., Kang, Y. M., Choi, J. Y., Hwang, D. H. and Kim, B. G. (2013). Contribution of macrophages to enhanced regenerative capacity of dorsal root ganglia sensory neurons by conditioning injury. J Neurosci 33(38): 15095-15108.
  3. Kwon, M. J., Shin, H. Y., Cui, Y., Kim, H., Thi, A. H., Choi, J. Y., Kim, E. Y., Hwang, D. H. and Kim, B. G. (2015). CCL2 mediumtes neuron-macrophage interactions to drive proregenerative macrophage activation following preconditioning injury. J Neurosci 35(48): 15934-15947.

简介

已知巨噬细胞在神经损伤后的轴突再生中发挥有益作用。 为了开发一种体外模型,其中损伤信号可以引发再生巨噬细胞活化,我们建立了由成体背根神经节感觉神经元和腹膜巨噬细胞组成的共培养物,并加入cAMP类似物二丁酰基cAMP。 从共同培养物收集的条件培养基表现出强烈的神经突生长活动。 通过添加米诺环素(巨噬细胞减活剂)几乎完全消除神经突生长活动,表明负责神经突生长的因子是由活化的巨噬细胞产生的。
【背景】成年哺乳动物的CNS神经元在损伤后不会自发再生轴突。 预处理周围神经损伤允许背根神经节(DRG)感觉轴突通过促进再生相关基因的表达来再生中心分支。 我们以前已经表明,预处理损伤后DRG中的活化巨噬细胞有助于提高DRG感觉神经元的内在再生能力(Kwon等,2013)。 为了确定参与神经损伤后巨噬细胞活化的分子因子,我们开发了体外模型,其中神经元 - 巨噬细胞相互作用由cAMP引起,cAMP是增强神经元再生能力的众所周知的试剂。 与使用酵母聚糖激活巨噬细胞的以前的模型相比,我们的模型在预处理外周损伤模型中使用类似于分子事件的更多的生理刺激。

材料和试剂

  1. 1.5 ml Eppendorf管
  2. 15 ml锥形管
  3. 具有0.4μm透明PET膜(Corning,Falcon ,目录号:353090)的细胞培养插入物
  4. 70μm尼龙细胞过滤器(Corning,Falcon ,目录号:352350)
  5. 50ml锥形管
  6. 6孔板
  7. 0.2μm滤光片(BD)
  8. 8孔培养载玻片(Corning,Biocoat TM ,目录号:354632)
  9. Dulbecco's改良的Eagle培养基(DMEM)(GE Healthcare,Hyclone ,目录号:SH30243.01)
  10. 来自Clost 溶组织um剂(Sigma-Aldrich,目录号:C9407-100MG)的胶原酶
  11. Neurobasal 培养基(Thermo Fisher Scientific,Gibco TM ,目录号:21103-049)
  12. B-27 补充(50x),无血清(Thermo Fisher Scientific,Gibco TM ,目录号:17504-044)
  13. GlutaMax TM (Thermo Fisher Scientific,Gibco TM ,目录号:35050-061)
  14. 青霉素 - 链霉素(10,000U/ml,10,000μg/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140-122)
  15. 聚-D-赖氨酸氢溴酸盐(Sigma-Aldrich,目录号:P6407-5MG)
  16. 层粘连蛋白(Thermo Fisher Scientific,Gibco TM ,目录号:23017-015)
  17. 磷酸盐缓冲盐水(PBS)(GE Healthcare,Hyclone ,目录号:SH30256.01)
  18. 红细胞裂解缓冲液(QIAGEN,目录号:158904)
  19. 10%FBS(GE Healthcare,Hyclone TM ,目录号:SH30919.03)
  20. 腺苷3',5'-环状单磷酸酯,N 6 6 - 叔丁基2'-丁基 - 钠盐(db-cAMP;100μM)(EMD Millipore,Calbiochem。 sup>®,目录号:28745)
  21. 米诺环素(Sigma-Aldrich,目录号:M9511)
  22. 多聚甲醛-13 C(Sigma-Aldrich,目录号:604380)
  23. 正常山羊血清(Thermo Fisher Scientific,Gibco TM ,目录号:16210072)
  24. Triton X-100(DAEJUNG CHEMICAL& MITALS,目录号:8566-4405)
  25. 山羊抗小鼠IgG(H + L)第二抗体,Alexa Fluor?594缀合物(Thermo Fisher Scientific,Invitrogen TM,目录号:A11005)
  26. 抗βIII微管蛋白(Tuj-1)(Promega,目录号:G7121)
  27. 神经元培养基(见配方)
  28. 巨噬细胞培养基(参见配方)

设备

  1. 细剪
  2. 细镊子
  3. 血细胞计数器(Marienfeld-Superior)
  4. 解剖立体显微镜(Carl Zeiss,型号:Stemi DV4)
  5. 细胞培养CO 2培养箱(Panasonic)
  6. 扭动振动器(FINEPCR,型号:Twist shaker Tw3t)
  7. 台式离心机(Sorvall)
  8. 移液助剂
  9. 共聚焦显微镜(Olympus,型号:IX71)

软件

  1. ImageJ( http://rsbweb.nih.gov/ij/index。 html

程序

  1. 成年DRG神经元获自成年C57BL6小鼠。在处死动物后,解剖皮肤和椎旁肌肉以暴露脊柱。从S1到C1水平的双侧背根神经节(DRG)使用细剪刀和钳子在解剖显微镜下新鲜地解剖并浸泡在新鲜的冷DMEM中。确保在从根和神经分离DRG时小心不要损伤DRG。
    注意:尽快解剖DRG非常重要。从一只鼠标收集的总DRGs不应超过30分钟。
  2. 转移DRGs到一个1.5毫升的Eppendorf管用蓝色吸头尖端切断。快速向下旋转DRG并从DMEM中吸出。加入1ml含有125U/ml XI型胶原酶的DMEM,并使用保持在培养箱中的旋转振荡器在37℃温育旋转90分钟。转速的最佳范围约为35-45 rpm。
  3. 吸出胶原酶的DMEM和重悬DRG与1毫升DMEM。当添加DMEM时,DRGs将浮动。等待DRGs下降,然后吸出。重复此步骤至少五次,以完全去除胶原酶。
    注意:尽量不要完全吸出。重要的是要防止DRGs被吸出。
  4. 使用截止的蓝色移液器吸头转移DRGs到15毫升锥形管。然后使用蓝色尖端研磨至少15次,直到获得均匀的细胞悬浮液 注意:研磨应该很温柔。避免产生气泡,尽量不要触及锥形管的底部。
  5. 在约239×g离心管3分钟以除去上清液中的细胞碎片。重悬细胞沉淀在1ml Neurobasal培养基补充B27和胰蛋白酶5至10次。然后使细胞通过安装在50ml锥形管上的70μm细胞过滤器。等待2分钟,然后使用吸管帮助过滤器倒入Neurobasal/B-27培养基,以便通过过滤器的细胞容易脱落。再等一分钟。
  6. 将板解离的细胞置于用0.01%聚-D-赖氨酸和层粘连蛋白(3μg/ml)预包被的6孔板上,并非常温和地摇动板以使细胞均匀分布。每孔加入总共6×10 6个DRG神经元。然后,将板置于保持在37℃的CO 2培养箱中 注意:用蒸馏水稀释聚-D-赖氨酸(0.01%)和层粘连蛋白(3μg/ml)。聚-D-赖氨酸在37℃包被平板2小时或在4℃过夜,然后用蒸馏水洗涤平板两次。然后,在室温下用层粘连蛋白包被平板2小时,并用PBS洗涤两次。然后在室温下干燥板,然后进行细胞接种。
  7. 共培养需要在解离的DRG神经元的初始铺板后4小时建立。原代腹膜巨噬细胞由成年C57BL6小鼠制备。在CO 2室中处死小鼠后,精细地解剖腹部皮肤以暴露腹膜。
  8. 注入10ml冰冷PBS到腹膜。轻轻按摩腹膜1-2分钟。然后,吸出PBS并转移到新的50ml锥形管。
  9. 将灌洗液在239℃下在4℃离心10分钟以沉淀细胞。将细胞沉淀物在室温(RT)下重悬于3ml的红细胞裂解缓冲液中3分钟,然后在4℃下以239×g离心10分钟以再次沉淀细胞。
  10. 将沉淀的细胞重悬浮于巨噬细胞培养基中。将腹膜巨噬细胞接种在放置在6孔板上的细胞培养插管上,其中在4小时前已经接种了解离的DRG神经元。铺板的巨噬细胞的数目保持为培养的DRG神经元的数目的5倍
  11. 巨噬细胞铺板后4小时,用二丁酰基-cAMP(db-cAMP;100μM)或PBS作为对照处理神经元 - 巨噬细胞共培养物。在一些孔中,巨噬细胞去活化剂米诺环素与db-cAMP以10μg/ml的浓度加入。
  12. 24小时后,用新鲜巨噬细胞培养基替换培养基。共培养物保持72小时而不更换培养基
  13. 然后收集条件培养基,在239×g离心5分钟,并通过0.2μm过滤器以除去任何残留的细胞碎片。将收集的条件培养基保存在-70℃直至使用。
  14. 对于神经突生长测定,使用步骤1-3中所述的相同方法获得成体DRG神经元并解离。
  15. 将细胞沉淀重悬于补充有B27的Neurobasal-A中,将总共1×10 4个细胞(每孔)接种在用0.01%聚-D-赖氨酸预包被的8孔培养载玻片上和3μg/ml层粘连蛋白。使用与步骤6中所述相同的方法进行预涂。
  16. 将神经元培养物在培养箱中保持2小时,使细胞附着于底部。并且用在步骤15中收集的解冻的条件培养基替换培养基
  17. 在初始铺板15小时后,用4%冷的多聚甲醛直接固定细胞20分钟。
  18. 载玻片上的固定细胞用1x PBS洗涤三次,并用由10%NGS和0.1%Triton-X组成的封闭溶液孵育30分钟。然后,用1x PBS洗涤两次,用10%NGS以1:1,000稀释在室温下4小时或在4℃过夜进行一抗(Tuj-1)染色。
  19. 用1×PBS洗涤两次,并且在室温下用10%NGS以1:500稀释进行2小时的第二抗体(山羊抗兔)染色。然后,用1x PBS洗涤载玻片两次,并用安装凝胶将盖玻片安装在载玻片上
  20. 使用共焦显微镜对免疫染色的βIII微管蛋白进行图像以显现神经突生长。

数据分析

  1. 测量每个神经元的平均神经突长度以比较由不同条件培养基诱导的神经突向外生长的程度。神经节长度使用用于图像分析软件套件NIH ImageJ的Neuron J插件测量(可从 http://rsbweb.nih.gov/ij/index.html ),这有助于细长结构的跟踪和量化。每个实验条件在每个培养物的两个孔中复制。将每个孔分成四个象限,在每个象限的中心获得200×放大倍数的图像(每个孔中有四个图像)。对每个图像中的所有神经元跟踪神经突,并且手动计数每个图像的DRG神经元的数量。观察到每个图像大约20个神经元,因此,对于每个孔测量约80个神经元。通过将总神经突长度除以神经元总数来计算每个孔中每个神经元的平均神经突长度。
  2. 从用PBS处理的神经元 - 巨噬细胞共培养物收集的条件培养基不支持神经突生长(图1A)。当DRG神经元与用二丁酰基cAMP处理的条件培养基一起生长时,它们不变显示出稳定的神经突生长(图1B)。如果二甲胺四环素,一种巨噬细胞去活化因子与cAMP一起被添加到神经元 - 巨噬细胞共培养物中,神经突生长活性几乎完全被消除,表明神经突生长活性需要巨噬细胞的激活(图1C)。在我们以前的研究中,我们显示从cAMP处理的神经元或单独的巨噬细胞培养物收集的条件培养基不支持神经突生长(Kwon等人,2013)。因此,神经元和巨噬细胞之间的相互作用对于神经突生长活性是至关重要的

    图1.神经元 - 巨噬细胞相互作用促进神经突生长活性。用PBS(A),cAMP(B)和cAMP +米诺环素(C)处理神经元(成年DRG神经元)和腹膜巨噬细胞共培养物, 。比例尺=100μm。

笔记

  1. 在使用这个协议的神经突生长测定中,DRG神经元在培养物的15小时内不生长任何显着的神经突。然而,如果允许DRG神经元生长超过15小时,即使在控制条件下也可以观察到一定程度的轴突生长。在先前研究中使用大鼠DRG神经元的神经突生长测定中报道了类似的发现(Cafferty等人,2004)。在试验实验中,我们测试几个不同的文化持续时间,我们发现15 h的文化导致控制条件最小的神经突生长,而用cAMP治疗的条件培养基实现了高度稳定的神经突生长。
  2. 为了建立神经元 - 巨噬细胞共培养物,可以将离解的巨噬细胞直接添加到没有细胞培养插入物的培养的DRG神经元中。在我们以前的研究中,我们直接比较了从直接共培养物收集的条件培养基和来自与通过细胞培养插入物分开的两种细胞类型的共培养物之间的神经突向外生长的程度(Kwon et al。 ,2013),两种条件之间没有差异。这表明两种细胞类型使用可溶性分子彼此连通。在我们的实验室中的以下研究发现,在这种共培养模型中,神经元分泌的CCL2负责将巨噬细胞活化为促再生表型(Kwon等人,2015)。

食谱

  1. 神经元培养基
    神经基础培养基(500ml) 2%B27
    1%glutamax
    1%青霉素 - 链霉素
  2. 巨噬细胞培养基
    DMEM
    10%FBS
    1%青霉素 - 链霉素

致谢

该协议由大韩民国科学,ICT和未来规划部的赠款NRF-2015R1A2A1A01003410支持。

参考文献

  1. Cafferty,WB,Gardiner,NJ,Das,P.,Qiu,J.,McMahon,SBand Thompson,SW(2004)。  调节损伤诱导的脊髓轴突再生在白细胞介素-6敲除小鼠中失败。 J Neurosci 24 ):4432-4443。
  2. 权利要求1所述的方法包括以下步骤:(a)提取一个或多个特征向量,其中h = "http://www.ncbi.nlm.nih.gov/pubmed/24048840"target ="_ blank">巨噬细胞通过调节损伤对背根神经节感觉神经元的再生能力的贡献。 Neurosci 33(38):15095-15108。
  3. (a)和(b)所示,我们的研究结果表明,这些研究结果表明,在预处理损伤后,插入文件"href ="http://www.ncbi.nlm.nih.gov/pubmed/26631474"target ="_ blank"> CCL2培养基神经元 - 巨噬细胞相互作用以驱动促再造巨噬细胞激活。 > J Neurosci 35(48):15934-15947。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
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. Yun, H. J. and Kim, B. S. (2016). An in vitro Model of Neuron-macrophage Interaction to Generate Macrophages with Neurite Outgrowth Properties. Bio-protocol 6(22): e2012. DOI: 10.21769/BioProtoc.2012.
  2. Kwon, M. J., Shin, H. Y., Cui, Y., Kim, H., Thi, A. H., Choi, J. Y., Kim, E. Y., Hwang, D. H. and Kim, B. G. (2015). CCL2 mediumtes neuron-macrophage interactions to drive proregenerative macrophage activation following preconditioning injury. J Neurosci 35(48): 15934-15947.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。