Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells
Neuro2a细胞中淀粉样前体蛋白（APP）的内吞回收测定
发布: 2017年12月05日第7卷第23期 DOI: 10.21769/BioProtoc.2635 浏览次数: 8268
评审: Anonymous reviewer(s)
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The authors used this protocol in:
Jan 2017
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Abstract
The established primary trigger of Alzheimer’s disease’s is β-amyloid (Aβ) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane (Koo et al., 1996; Ubelmann et al., 2017). The following recycling assay has been optimized to assess APP recycling by live murine Neuro2a cells, a neuroblastoma cell line (Ubelmann et al., 2017).
Keywords: APP (APP)
Recycling (回收)
Alzheimer’s (阿尔茨海默病)
Immunofluorescence (免疫荧光)
Background
Aβ42 accumulation is a primary trigger of Alzheimer’s disease. APP endocytosis is required for Aβ42 generation (Koo and Squazzo, 1994; Grbovic et al., 2003; Cirrito et al., 2008; Rajendran et al., 2008). Upon endocytosis, APP can recycle back to the plasma membrane likely escaping processing at endosomes. While many studies have characterized APP endocytosis, mechanisms that regulate APP recycling need to be established. A robust, sensitive and quantitative assay is thus necessary. APP recycling has been assessed qualitatively by immunofluorescence and quantitatively using bulk biotinylation of surface proteins followed by a chase for endocytosis and a chase for recycling after stripping or blocking of surface proteins (Koo et al., 1996; Yamazaki et al., 1996; Chaufty et al., 2012).
We developed a methodology for following and measuring APP recycling in Neuro2a cells using classical immunofluorescence and semi-quantitative cell biology methods. Our assay relies on the transient expression of APP tagged with the red fluorescent protein (RFP), to detect and quantify the cellular pool of APP and on using an antibody against the ectodomain of APP, to selectively detect and quantify the trafficking of the surface pool of APP.
Materials and Reagents
- Circular glass coverslips, 13 mm (VWR, Marienfeld, catalog number: 630-1597 )
Note: Autoclaved, pre-washed with 40% ethanol/60% HCl 1 h at RT and washed 4 times, 15 min each, with Milli-Q water at RT.
- Superfrost glass slides (MENZEL GERHARD, catalog number: 2586E )
- 24-well dishes (SARSTEDT, catalog number: 83.1836 ) for mammalian cell culture
- Plastic Pasteur pipette (SARSTEDT, catalog number: 86.1171 )
- Parafilm (Fisher Scientific, catalog number: 11782644)
Manufacturer: Bemis, Parafilm, catalog number: PM999 .
- Neuroblastoma Neuro2a cells (ATCC, catalog number: CCL-131 )
- APP-RFP plasmid (Szodorai et al., 2009) (S. Kins, University of Kaiserslautern)
- Culture medium: Dulbecco’s modified Eagle medium (DMEM)-GlutaMAX (Thermo Fisher Scientific, GibcoTM, catalog number: 10566016 ) with 10% fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F0804 ); 100 U/ml penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
- Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
- Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
- Lipofectamine RNAiMax (Thermo Fisher Scientific, InvitrogenTM, catalog number: 13778150 )
- Murine Anti-APP N-terminal monoclonal antibody (22C11) (Merck, catalog number: MAB348 )
- HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
- Phosphate buffer saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010031 )
- Donkey anti-mouse Alexa 488 (Thermo Fisher Scientific, catalog number: A-21202 )
- Coverslip-slide Mounting solution (FluoroMount-G) (Southern Biotech, catalog number: 0100-01 )
- Saponin (Sigma-Aldrich, catalog number: 47036 )
- Sodium chloride (NaCl) (NZYTech, catalog number: MB15901 )
- Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
- Acetic acid (Merck, catalog number: 1.00063.2511 )
- Acid-stripping buffer (see Recipes)
- Fixative solution (see Recipes)
Equipment
- CO2 incubator for primary cell culture (BINDER, model: CB 160 )
- Counting chamber (Belden, Hirschmann, catalog number: 8100103 )
- Epifluorescence upright microscope Z2 (Carl Zeiss, model: Axio Imager Z2 ) equipped a 60x NA-1.4 oil immersion objective and an AxioCam MRm CCD camera (Carl Zeiss)
Software
- ImageJ 2.0 v1.51n software (free download from http://rsb.info.nih.gov/ij/)
- GraphPad Prism 6 (https://www.graphpad.com/scientific-software/prism/)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Ubelmann, F., Burrinha, T. and Guimas Almeida, C. (2017). Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells. Bio-protocol 7(23): e2635. DOI: 10.21769/BioProtoc.2635.
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分类
免疫学 > 免疫细胞成像 > 荧光显微技术
细胞生物学 > 细胞成像 > 荧光
生物化学 > 蛋白质 > 免疫检测 > 免疫染色法
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