Primary Explosive Blast-induced Traumatic Brain Injury Model in PC12 Cell Culture
PC12 细胞培养中建立的爆炸冲击诱导的初级脑损伤模型
发布: 2016年08月20日第6卷第16期 DOI: 10.21769/BioProtoc.1907 浏览次数: 7155
评审: Soyun KimPengpeng LiAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Sep 2015
Abstract
While it is understood that structural damage occurs at the cellular level from the traumatic brain injury event, the effect on functional activity remains largely unknown. Simplified models such as in vitro models of primary explosive blast are critically needed to deconvolute mechanisms of cellular damage. This protocol details an in vitro indoor experimental system setup (Zander et al., 2015) using real military explosive charges to more accurately represent battlefield blast exposure, and probe the effects of primary explosive blast on dissociated neurons.
Keywords: Primary explosive blast model (初级炸药爆炸模型)
Blast induced traumatic brain injury model (创伤性脑损伤诱导的爆炸模型)
Cell culture (细胞培养)
Materials and Reagents
- 12 mm circular glass coverslips (Fisher Scientific, catalog number: 12-545-80 )
- 24-well plates (Corning, FalconTM, catalog number: 351147 )
- Sterile SealPlate® covers (Excel Scientific)
- PC12 cells (ATCC, catalog number: CRL-1721 )
- Mouse laminin (Corning, catalog number: 354239 )
- 100% ethanol
- Polylysine (Sigma-Aldrich, catalog number: P5899 )
- RPMI 1640 (Corning, catalog number: 10-041-CV )
- Horse serum (Corning, catalog number: 35-030-CV )
- Fetal bovine serum (FBS) (Fisher Scientific, catalog number: SH3007003IH )
- Dulbecco Modified Eagle’s Medium (DMEM) (Corning, catalog number: 10-013-CV )
- Calf serum (GE Healthcare Life Sciences, HycloneTM, catalog number: SH30072.03 )
- Nerve growth factor (NGF) (Corning, catalog number: 356004 )
- Spherical 1.77 grams/cm3 cyclotrimethylene trinitramine Class 5 (RDX Class V) charges
- HEPES (Fisher Scientific, catalog number: BP299500 )
- 10-gallon poly (methyl methacrylate) (PMMA)
- Calcein-AM (Thermo Fisher Scientific, catalog number: C3099 )
- Ethidium homodimer-1 (Thermo Fisher Scientific, catalog number: E1169 )
- Phosphate buffered saline (PBS) (Fisher Scientific, catalog number: BP399500 )
- Lactate dehydrogenase cytotoxicity assay kit (LDH) (Abnova, catalog number: KA0785 )
- RIPA buffer (Alfa Aesar, catalog number: J62189-AP )
- Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
- The micro BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23235 )
- Hank’s Balanced Salt Solution (HBSS) (Corning, catalog number: 21-022-CV )
- Calcein (MP Biomedicals, catalog number: 190167 )
- Penicillin/Streptomycin, 50x (Corning, catalog number: 30-001-Cl )
- Antibiotic/antimycotic (see Recipes)
- Growth/complete medium (see Recipes)
- Differentiation medium (see Recipes)
Equipment
- Incubator
- Aquarium
- Three piezoelectric high frequency dynamic pressure sensors (PCB Piezotronics, model: 102A )
- Sterile hood
- Oven
- Zeiss LSM5 Pascal equipped with Epiplan-Neofluar lenses
- Confocal laser scanning microscopy (CLSM)
Software
- ImageJ v 1.34
- Zeiss LSM software (v 4.2.0.121)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Piehler, T., Zander, N. and Benjamin, R. (2016). Primary Explosive Blast-induced Traumatic Brain Injury Model in PC12 Cell Culture. Bio-protocol 6(16): e1907. DOI: 10.21769/BioProtoc.1907.
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分类
神经科学 > 行为神经科学 > 认知
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