Epidermal Growth Factor (EGF) Receptor Endocytosis Assay in A549 Cells
A549 细胞中的表皮生长因子（GCF）受体内吞作用分析
发布: 2013年08月05日第3卷第15期 DOI: 10.21769/BioProtoc.847 浏览次数: 19497
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The authors used this protocol in:
Nov 2012
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Abstract
The following endocytosis assay has been optimized to assess EGF-stimulated EGFR endocytosis; but could be modified to assess other ligand-stimulated endocytosis of plasma membrane receptors (for which fluorochrome-conjugated ligands are available to track their receptor internalization). In brief, cells are treated with fluorescent EGF at 4 °C to allow binding to the receptor, but not internalization; then, endocytosis is allowed at 37 °C for different timepoints. For the setting up of this protocol we are really indebted to Dr. Letizia Lanzetti (Lanzetti et al., 2000).
Keywords: Internalization (内化)
Signaling (信号)
Egfr (表皮生长因子受体)
Mammalian cells (哺乳动物细胞)
Fluorescence (荧光)
Materials and Reagents
- A549 non-small lung carcinoma cells (ATCC)
- D-MEM medium (Sigma-Aldrich)
- 10% serum RPMI medium (Sigma-Aldrich)
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
- Fluorescent EGF-555 (Life Technologies, InvitrogenTM, catalog number: E35350 )
- 100% Acetic acid
- 5 M NaCl solution
- Paraformaldehyde (Fluka)
- Phosphate buffer saline (PBS)
- Coverslip-Slide Mounting solution (Fluoromonut, Southern Biotech)
- 4',6-diamidino-2-phenylindole (DAPI) (F. Hoffmann-La Roche)
- Saponin (Sigma-Aldrich, catalog number: S4521 )
- Acid wash (see Recipes)
- 4% PAF in PBS (see Recipes)
Equipment
- Round glass cover slips
- 6-well and 24-well dishes for mammalian cell culture (Corning, Costar®)
- Lipofectamine2000 (Life Technologies, InvitrogenTM)
- Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems)
- 4 °C refrigerator
- Cell culture incubator humidified 5% CO2 atmosphere
- pH-meter
Software
- ImageJ software (free download from http://rsb.info.nih.gov/ij/)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Rizzolio, S. and Tamagnone, L. (2013). Epidermal Growth Factor (EGF) Receptor Endocytosis Assay in A549 Cells. Bio-protocol 3(15): e847. DOI: 10.21769/BioProtoc.847.
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分类
细胞生物学 > 细胞成像 > 荧光
细胞生物学 > 基于细胞的分析方法 > 细胞吞排作用 > 内吞作用
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