Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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

FU Florent Ubelmann
Tatiana Burrinha Tatiana Burrinha
Claudia Guimas Almeida Claudia Guimas Almeida

发布: 2017年12月05日第7卷第23期 DOI: 10.21769/BioProtoc.2635 浏览次数: 9053

评审: Anonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of EMBO Reports, featuring study using the protocol.

Jan 2017

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

利用带网格培养皿实现活细胞延时成像到免疫荧光成像的衔接观察

利用带网格培养皿实现活细胞延时成像到免疫荧光成像的衔接观察

Nick Lang [...] Andrew D. Stephens

2026年02月20日 1316 阅读

基于免疫荧光的骨髓活检样本原位 mRNA 表达与蛋白同步检测

基于免疫荧光的骨髓活检样本原位 mRNA 表达与蛋白同步检测

Alba Lillo Sierras [...] Stefan Scheding

2026年06月20日 1227 阅读

利用过氧化物酶融合纳米抗体和荧光化酪酰胺-葡萄糖氧化酶反应检测 1 mm 厚小鼠脑切片中的靶分子

利用过氧化物酶融合纳米抗体和荧光化酪酰胺-葡萄糖氧化酶反应检测 1 mm 厚小鼠脑切片中的靶分子

Kenta Yamauchi [...] Hiroyuki Hioki

2026年06月05日 725 阅读

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) search Recycling (回收) search Alzheimer’s (阿尔茨海默病) search Immunofluorescence (免疫荧光) search

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

  1. 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.
  2. Superfrost glass slides (MENZEL GERHARD, catalog number: 2586E )
  3. 24-well dishes (SARSTEDT, catalog number: 83.1836 ) for mammalian cell culture
  4. Plastic Pasteur pipette (SARSTEDT, catalog number: 86.1171 )
  5. Parafilm (Fisher Scientific, catalog number: 11782644)
    Manufacturer: Bemis, Parafilm, catalog number: PM999 .
  6. Neuroblastoma Neuro2a cells (ATCC, catalog number: CCL-131 )
  7. APP-RFP plasmid (Szodorai et al., 2009) (S. Kins, University of Kaiserslautern)
  8. 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 )
  9. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  10. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
  11. Lipofectamine RNAiMax (Thermo Fisher Scientific, InvitrogenTM, catalog number: 13778150 )
  12. Murine Anti-APP N-terminal monoclonal antibody (22C11) (Merck, catalog number: MAB348 )
  13. HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  14. Phosphate buffer saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010031 )
  15. Donkey anti-mouse Alexa 488 (Thermo Fisher Scientific, catalog number: A-21202 )
  16. Coverslip-slide Mounting solution (FluoroMount-G) (Southern Biotech, catalog number: 0100-01 )
  17. Saponin (Sigma-Aldrich, catalog number: 47036 )
  18. Sodium chloride (NaCl) (NZYTech, catalog number: MB15901 )
  19. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  20. Acetic acid (Merck, catalog number: 1.00063.2511 )
  21. Acid-stripping buffer (see Recipes)
  22. Fixative solution (see Recipes)

Equipment

  1. CO2 incubator for primary cell culture (BINDER, model: CB 160 )
  2. Counting chamber (Belden, Hirschmann, catalog number: 8100103 )
  3. 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

  1. ImageJ 2.0 v1.51n software (free download from http://rsb.info.nih.gov/ij/)
  2. GraphPad Prism 6 (https://www.graphpad.com/scientific-software/prism/)

Procedure

English
中文翻译

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.

ris ris Download Citation in RIS Format

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

您对这篇实验方案有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件