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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   

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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

  1. A549 non-small lung carcinoma cells (ATCC)
  2. D-MEM medium (Sigma-Aldrich)
  3. 10% serum RPMI medium (Sigma-Aldrich)
  4. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
  5. Fluorescent EGF-555 (Life Technologies, InvitrogenTM, catalog number: E35350 )
  6. 100% Acetic acid
  7. 5 M NaCl solution
  8. Paraformaldehyde (Fluka)
  9. Phosphate buffer saline (PBS)
  10. Coverslip-Slide Mounting solution (Fluoromonut, Southern Biotech)
  11. 4',6-diamidino-2-phenylindole (DAPI) (F. Hoffmann-La Roche)
  12. Saponin (Sigma-Aldrich, catalog number: S4521 )
  13. Acid wash (see Recipes)
  14. 4% PAF in PBS (see Recipes)

Equipment

  1. Round glass cover slips
  2. 6-well and 24-well dishes for mammalian cell culture (Corning, Costar®)
  3. Lipofectamine2000 (Life Technologies, InvitrogenTM)
  4. Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems)
  5. 4 °C refrigerator
  6. Cell culture incubator humidified 5% CO2 atmosphere
  7. pH-meter

Software

  1. ImageJ software (free download from http://rsb.info.nih.gov/ij/)

Procedure

  1. Day 1
    Seed the appropriate number of cells (see Note 2) on top of glass coverslips placed (one per well) in 24-well cell culture dishes. Alternatively, if it is required to transfect cells prior to performing the endocytosis assay, seed cells on top of 5-6 coverslips placed in each well of 6-well culture dishes. You should prepare at least one independent multi-well dish per each time point in your experiment, plus two for controls (e.g. four dishes for an experiment with control cells plus two different times of endocytosis).
  2. Day 2 (optional)
    Cell DNA-transfection with Lipofectamine2000, according to manufacturer’s protocol. On the day of the endocytosis assay, transfer the coverslips with cells into 24-well cell culture dishes. Transfection could be needed when you need to modulate proteins putatively involved in the internalization of the receptor.
  3. Day 3-4
    Endocytosis assay (or Day 2-3, if no cell transfection needed prior to the assay), by the following steps (see Figure 1 for the protocol scheme).


    Figure 1. Schematic representation of experimental flow

    1. Remove cell culture medium and incubate cells with 400-500 μl of serum-free DMEM medium containing 0.3% BSA for at least 1 h (extendable up to 3 h) at 37 °C in cell culture incubator, in order to deprive cells of serum-contained stimuli.
    2. Replace cell medium with the appropriate dilution of the fluorescent ligand EGF (2 ng/ml) into 400 μl of DMEM 0.3% BSA (per well).
    3. Incubate the cells on ice for 1 h, in the dark (by covering the dish with aluminium foil).
    4. Wash two multi-well dishes with ice-cold PBS three times and leave them on ice (control condition = ligand binding without endocytosis, at 4 °C binding ligand receptor is allowed, while internalization is blocked).
    5. On one of the two dishes above, perform an acidic wash (to remove non-internalized ligand) by incubating the cells with an acetic acid buffer on ice for 6 min. Rinse with ice-cold PBS three times and leave on ice.
    6. Transfer the other cell culture dishes at 37 °C to elicit endocytosis for different periods (optimal time points need to be set for the specific cellular model and ligand stimulation; in our experience, for example, 12 and 25 min for EGF 2 ng/ml to A549 cells).
    7. At the end of each incubation, block endocytosis by moving dishes on ice.
    8. Wash 3 times with ice-cold PBS and proceed with acid wash on ice as described in step 5 (to detach all the ligand bound to the receptor at the plasma membrane and allow detection of internalized ligand only).
    9. After acidic wash, rinse each well 3 times with cold PBS.
    10. Fix cells on every coverslip by incubation with 4% PFA in PBS for 10 min at room temperature and then wash 3 times with PBS. Fixed cells may be stored for several days in the refrigerator at this stage.
    11. Permeabilize cell membranes by incubating fixed cells on coverslips with a solution of 0.01% saponin in PBS 1% BSA for 5-10 min on ice (in alternative, you may use 0.1% Triton in PBS 1% BSA).
    12. Wash permeabilized cells three times with PBS.
    13. (Optional) If Immunofluorescence analysis (IFA) is planned, non-specific binding may be blocked by incubation with PBS 1% BSA for 30 min, followed by immunostaining with specific antibodies.
    14. Counterstain cell nuclei by incubating with DAPI 5 min at room temperature (a dilution 1:10,000 of a stock of 1 μg/μl).
    15. Finally, wash once coverslips in PBS, then rinse with water and fix them inverted on microscope glass slides by including a mounting medium.
    16. Analysis of fluorescent signals should be done with a confocal microscope with a 63x magnification, such as Leica TCS SP2 AOBS confocal laser-scanning microscope (see Figure 2 for representative images).
    17. Quantify endocytosed fluorescent signal acquired with ImageJ software.


      Figure 2. Representative pictures of endocytosis assay on A549. On the left, control cells treated with fluorescent EGF 1 h at 4 °C and subjected to acidic wash to clear out all EGF bound to plasmamembrane; no fluorescent signal from the plasmamembrane. On the right, cells treated with fluorescent EGF 1 h at 4 °C, subjected to acidic wash and then shifted to 37 °C (internalization); fluorescent signal from internalized EGF.

Notes

  1. I settled this protocol on A549 cells, other cell lines may require optimization of experimental conditions (such as different timing of endocytosis induction at 37 °C). A549 should be grown in 10% serum RPMI medium.
  2. Grow cells on coverslips in their optimal culture medium to reach 50-80% confluency on the day of the assay. Remember to have in separate multiwell dishes the coverslips that will have to undergo the different incubation times or control conditions.
  3. In the meanwhile of step 2 (cell incubation with 0.3% BSA solution), prepare fluorochrome-conjugated ligand solution at the desired final concentration in DMEM 0.3% BSA, considering 400-500 μl per each well (in 24-well dishes). Remember to keep ligand-containing solutions always in the dark (covered with aluminium foil).
  4. Put a plastic box (large enough to contain all multiwell dishes) with one inch of water on the bottom to warm up at 37 °C in the cell incubator that you will use for the assay. This will serve to speed up the temperature change between 4 °C and 37 °C to allow accurate timing for endocytosis in the different conditions.
  5. Pay attention not to put the acidic wash solution directly on cells to avoid causing their detachment, but instead gently pour it on the side of the wells.
  6. Do not exceed 6 min incubation with acidic wash. Acidic wash for coverslips always kept on ice, will provide the negative control of the experiments: In fact, after acidic wash, you should not detect any fluorescent signal from the ligand associated with the cells. On the other hand, there should still be strong signal on PBS-washed cells not subjected to acidic wash, indicating that ligand binding to the receptor indeed occurred.
  7. It is good rule to acquire at least 50 different microscopic fields per each condition, from at least three independent experiments (applying constant detection parameters at the confocal microscope). Mean fluorescence intensity of the fields should then be quantified from image files (e.g. by ImageJ software).

Recipes

  1. Acid wash
    0.2 M acetic acid
    0.5 M NaCl (pH 2.8)
    For 100 ml
    1.2 ml 100% acetic acid
    10 ml 5 M NaCl
    88.8 ml H2O
  2. 4% PFA in PBS
    4 g paraformaldehyde in 100 ml PBS
    Heat PBS (shaking) for 30 min until it reaches 60 °C
    Add paraformaldehyde
    Clarify by dropwise addiction of 5 M NaOH
    Cool down temperature while shaking
    Adjust pH at 7.4 (with 5 M NaOH/6 N HCl)
    Bring to final volume with PBS
    Shake again the solution
    Aliquote
    Keep at -20 °C

Acknowledgments

This protocol was initially developed for the study Rizzolio et al. (2012). Previous reports analyzing receptors internalization that have been used as reference to set up this procedure include: Lanzetti et al. (2000); Sawamiphak et al. (2010) and Popovic et al. (2012). Correlated research activity in the authors’ lab was funded by the University of Torino-Compagnia di San Paolo, Grant ORTO11RKTW (RETHE). The authors are indebted to Dr. Letizia Lanzetti (University of Torino) for her expert support in setting up this protocol.

References

  1. Lanzetti, L., Rybin, V., Malabarba, M. G., Christoforidis, S., Scita, G., Zerial, M. and Di Fiore, P. P. (2000). The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5. Nature 408(6810): 374-377.
  2. Popovic, D., Akutsu, M., Novak, I., Harper, J. W., Behrends, C. and Dikic, I. (2012). Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers. Mol Cell Biol 32(9): 1733-1744.
  3. Rizzolio, S., Rabinowicz, N., Rainero, E., Lanzetti, L., Serini, G., Norman, J., Neufeld, G. and Tamagnone, L. (2012). Neuropilin-1-dependent regulation of EGF-receptor signaling. Cancer Res 72(22): 5801-5811. 
  4. Sawamiphak, S., Seidel, S., Essmann, C. L., Wilkinson, G. A., Pitulescu, M. E., Acker, T. and Acker-Palmer, A. (2010). Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis. Nature 465(7297): 487-491.

简介

以下内吞测定已经被优化以评估EGF刺激的EGFR内吞作用; 但可以修改以评估其他配体刺激的细胞膜受体的内吞作用(其中荧光染料偶联的配体可用于追踪它们的受体内化)。 简言之,在4℃下用荧光EGF处理细胞以允许结合受体,但不内化; 然后,在37℃下对不同的时间点进行内吞作用。 为了建立该方案,我们真正感谢Letizia Lanzetti博士(Lanzetti等人,2000)。

关键字:内化, 信号, 表皮生长因子受体, 哺乳动物细胞, 荧光

材料和试剂

  1. A549非小细胞肺癌细胞(ATCC)
  2. D-MEM培养基(Sigma-Aldrich)
  3. 10%血清RPMI培养基(Sigma-Aldrich)
  4. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3912)
  5. 荧光EGF-555(Life Technologies,Invitrogen TM ,目录号:E35350)
  6. 100%乙酸
  7. 5 M NaCl溶液
  8. 多聚甲醛(Fluka)
  9. 磷酸盐缓冲液(PBS)
  10. 盖玻片安装溶液(Fluoromonut,Southern Biotech)
  11. 4',6-二脒基-2-苯基吲哚(DAPI)(F.Hoffmann-La Roche)
  12. 皂苷(Sigma-Aldrich,目录号:S4521)
  13. 酸洗(见配方)
  14. PBS中的4%PAF(参见配方)

设备

  1. 圆形玻璃盖玻片
  2. 用于哺乳动物细胞培养的6孔和24孔培养皿(Corning,Costar )
  3. Lipofectamine 2000(Life Technologies,Invitrogen TM
  4. Leica TCS SP2 AOBS共聚焦激光扫描显微镜(Leica Microsystems)
  5. 4°C冰箱
  6. 细胞培养箱加湿5%CO 2气氛
  7. pH计

软件

  1. ImageJ软件(可从 http://rsb.info.nih.gov/ij/下载) )

程序

  1. 第1天
    在24孔细胞培养皿中放置的玻璃盖玻片(每孔一个)的顶部上种适当数量的细胞(参见注释2)。或者,如果需要在进行胞吞测定之前转染细胞,将5-6盖玻片顶部的种子细胞置于6孔培养皿的每个孔中。您应该为实验中的每个时间点准备至少一个独立的多孔板,另外两个用于对照(例如四个皿用于对照细胞加两个不同的内吞作用的实验)。
  2. 第2天(可选)
    根据制造商的方案用Lipofectamine2000进行细胞DNA转染。在内吞测定的当天,将盖玻片与细胞转移到24孔细胞培养皿中。当需要调节假定参与受体内化的蛋白质时,可能需要转染
  3. 第3-4天
    胞吞作用测定(或第2-3天,如果在测定之前不需要细胞转染),通过以下步骤(参见图1的方案方案)。


    图1.实验流程示意图

    1. 去除细胞培养基和细胞与400-500微升含有0.3%BSA的无血清DMEM培养基孵育至少1小时(可延长到3小时)在37℃在细胞培养孵化器,以剥夺细胞的血清 包含刺激。
    2. 用适当稀释的荧光配体EGF(2ng/ml)将细胞培养基更换为400μl的DMEM 0.3%BSA(每孔)。
    3. 孵育细胞在冰上1小时,在黑暗中(通过用铝箔覆盖菜)
    4. 用冰冷的PBS洗涤两个多孔板三次,并将它们留在冰上(对照条件=没有内吞的配体结合,在4℃下允许结合配体受体,而内化被阻断)。
    5. 在上述两个皿中的一个上,通过在冰上用乙酸缓冲液孵育细胞6分钟,进行酸洗涤(以除去非内化配体)。用冰冷的PBS冲洗三次,并放在冰上
    6. 转移其他细胞培养皿在37°C引起内吞作用不同的时期(最佳时间点需要设置为特定的细胞模型和配体刺激;在我们的经验,例如,12和25分钟为EGF 2 ng/ml到A549细胞)。
    7. 在每次孵育结束时,通过在冰上移动培养皿来阻断内吞作用。
    8. 用冰冷的PBS洗涤3次,并如步骤5所述在冰上进行酸洗(以分离在质膜上结合受体的所有配体,并仅允许检测内化配体)。
    9. 酸洗后,用冷PBS冲洗每个孔3次
    10. 通过在室温下用PBS中的4%PFA孵育10分钟将细胞固定在每个盖玻片上,然后用PBS洗涤3次。 固定电池可以在这个阶段在冰箱中存储几天
    11. 通过在冰上用0.01%皂苷在PBS 1%BSA中的溶液孵育盖玻片上的固定细胞5-10分钟(可选地,可以使用0.1%Triton的PBS 1%BSA)来透化细胞膜。
    12. 用PBS冲洗透化细胞三次
    13. (可选)如果计划进行免疫荧光分析(IFA),可以通过用PBS 1%BSA孵育30分钟,然后用特异性抗体免疫染色来阻断非特异性结合。
    14. 通过在室温下与DAPI温育5分钟(稀释度为1:10,000的1μg/μl的原液)复染细胞核。
    15. 最后,在PBS中清洗一次盖玻片,然后用水冲洗,并通过包括固定介质将它们倒置在显微镜载玻片上。
    16. 荧光信号的分析应该使用具有63x放大倍率的共聚焦显微镜,例如Leica TCS SP2 AOBS共聚焦激光扫描显微镜(参见图2的代表性图像)。
    17. 定量用ImageJ软件获得的内吞荧光信号

      图2.在A549上的胞吞作用测定的代表性图片。在左边,在4℃下用荧光EGF处理的对照细胞1小时,  进行酸洗以清除结合至质膜的所有EGF; 没有来自等离子膜的荧光信号。在右边,细胞 在4℃下用荧光EGF处理1小时,进行酸洗和 然后转移到37℃(内化);荧光信号 内化EGF。

笔记

  1. 我将这个协议安置在A549细胞上,其他细胞系可能需要优化实验条件(例如在37℃下不同的内吞诱导时间)。 A549应在10%血清RPMI培养基中生长
  2. 在其最佳培养基中在盖玻片上生长细胞,以在测定当天达到50-80%融合。 记住在单独的多孔菜肴中,盖玻片必须经历不同的孵育时间或控制条件
  3. 在步骤2(使用0.3%BSA溶液的细胞温育)的同时,考虑每孔(在24孔培养皿中)400-500μl,在DMEM 0.3%BSA中制备所需最终浓度的荧光染料缀合的配体溶液。 记住要保持配体溶液总是在黑暗中(用铝箔覆盖)
  4. 放置一个塑料盒(足够大,包含所有多孔菜)与一英寸的水在底部,在37℃在您将用于测定的细胞孵化器中预热。这将有助于加速在4°C和37°C之间的温度变化,以允许在不同条件下的内吞的准确时间。
  5. 注意不要将酸性洗涤溶液直接放在细胞上,以避免引起其分离,而是轻轻地将其倾倒在孔的侧面。
  6. 不要超过6分钟酸性洗涤孵育。酸性洗涤对于盖玻片总是保持在冰上,将提供实验的阴性对照:实际上,在酸性洗涤后,不应检测来自与细胞相关的配体的任何荧光信号。另一方面,在PBS洗涤的细胞上仍然存在未进行酸洗涤的强信号,表明配体与受体的结合确实发生了。
  7. 从每个条件获得至少三个独立实验(在共焦显微镜上应用恒定检测参数)获得至少50个不同的微观视野是有利的。然后应该从图像文件中量化场的平均荧光强度(例如通过ImageJ软件)。

食谱

  1. 酸洗
    0.2M乙酸
    0.5 M NaCl(pH 2.8)
    对于100毫升
    1.2ml 100%乙酸
    10ml 5M NaCl
    88.8ml H 2 O v/v
  2. 4%PFA的PBS溶液
    4g多聚甲醛在100ml PBS中的溶液 加热PBS(摇动)30分钟,直到它达到60℃
    加入多聚甲醛
    通过逐滴加入5M NaOH澄清 摇晃时冷却温度
    将pH调节至7.4(用5M NaOH/6N HCl)
    使用PBS
    达到最终体积 再次摇动解决方案
    等分
    保持在-20°C

致谢

该方案最初是为了研究Rizzolio等人(2012)开发的。 已经用作参考以建立该程序的分析受体内化的以前报道包括:Lanzetti等人(2000); Sawamiphak等人 。 (2010)和Popovic等人。 (2012)。 作者实验室的相关研究活动由都灵大学圣保罗大学,Grant ORTO11RKTW(RETHE)资助。 作者感谢Letizia Lanzetti博士(都灵大学)在设立此协议方面的专家支持。

参考文献

  1. Lanzetti,L.,Rybin,V.,Malabarba,M.G.,Christoforidis,S.,Scita,G.,Zerial,M.and Di Fiore,P.P。(2000)。 Eps8蛋白协调EGF 通过Rac的受体信号传导和通过Rab5的运输。 Nature 408(6810):374-377。
  2. Popovic,D.,Akutsu,M.,Novak,I.,Harper,J.W.,Behrends,C.and Dikic,I。(2012)。 自噬中的Rab GTPase激活蛋白:通过直接结合人ATG8修饰因子来调节内吞和自噬途径。 Mol Cell Biol 32(9):1733-1744。
  3. Rizzolio,S.,Rabinowicz,N.,Rainero,E.,Lanzetti,L.,Serini,G.,Norman,J.,Neufeld,G.and Tamagnone,L。(2012)。 神经毡蛋白1依赖性调节EGF受体信号传导。癌症研究 72(22):5801-5811。 
  4. Sawamiphak,S.,Seidel,S.,Essmann,C.L.,Wilkinson,G.A.,Pitulescu,M.E.,Acker,T.and Acker-Palmer,A。(2010)。 Ephrin-B2调节VEGFR2在发育和肿瘤血管生成中的功能。 465(7297):487-491。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
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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