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
发布: 2016年11月20日第6卷第22期 DOI: 10.21769/BioProtoc.2012 浏览次数: 8338
评审: Soyun KimAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2015
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
- 1.5 ml Eppendorf tubes
- 15 ml conical tube
- Cell culture insert with 0.4 μm transparent PET membrane (Corning, Falcon®, catalog number: 353090 )
- 70 μm nylon cell strainer (Corning, Falcon®, catalog number: 352350 )
- 50 ml conical tube
- 6 well plate
- 0.2 μm filter (BD)
- 8-well culture slide (Corning, BiocoatTM, catalog number: 354632 )
- Dulbecco’s modified Eagle medium (DMEM) (GE Healthcare, HycloneTM, catalog number: SH30243.01 )
- Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C9407-100MG )
- Neurobasal® medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103-049 )
- B-27® supplement (50x), serum free (Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044 )
- GlutaMaxTM (Thermo Fisher Scientific, GibcoTM, catalog number: 35050-061 )
- Penicillin-streptomycin (10,000 U/ml, 10,000 µg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
- Poly-D-lysine hydrobromide (Sigma-Aldrich, catalog number: P6407-5MG )
- Laminin (Thermo Fisher Scientific, GibcoTM, catalog number: 23017-015 )
- Phosphate-buffered saline (PBS) (GE Healthcare, HycloneTM, catalog number: SH30256.01 )
- Red blood cell lysis buffer (QIAGEN, catalog number: 158904 )
- 10% FBS (GE Healthcare, HycloneTM, catalog number: SH30919.03 )
- Adenosine 3’,5’-cyclic monophosphate, N6,O2’-dibutyryl-, sodium salt (db-cAMP; 100 μM) (EMD Millipore, Calbiochem®, catalog number: 28745 )
- Minocycline (Sigma-Aldrich, catalog number: M9511 )
- Paraformaldehyde-13C (Sigma-Aldrich, catalog number: 604380 )
- Normal goat serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16210072 )
- Triton X-100 (DAEJUNG CHEMICAL & MITALS, catalog number: 8566-4405 )
- Goat anti-mouse IgG (H + L) secondary antibody, Alexa Fluor® 594 conjugate (Thermo Fisher Scientific, InvitrogenTM, catalog number: A11005 )
- Anti β III tubulin (Tuj-1) (Promega, catalog number: G7121 )
- Neuron culture medium (see Recipes)
- Macrophage culture medium (see Recipes)
Equipment
- Fine scissors
- Fine forceps
- Hemacytometer (Marienfeld-Superior)
- Dissecting stereomicroscope (Carl Zeiss, model: Stemi DV4 )
- Cell culture CO2 incubator (Panasonic)
- Twist shaker (FINEPCR, model: Twist shaker Tw3t )
- Tabletop centrifuge (Sorvall)
- Pipette-aid
- Confocal microscope (Olympus, model: IX71 )
Software
- ImageJ (http://rsbweb.nih.gov/ij/index.html)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- 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.
- 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.
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分类
神经科学 > 细胞机理 > 细胞分离和培养
细胞生物学 > 细胞分离和培养
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