欢迎您, 登录 | 注册

首页 | English

X
加载中

The lipophilic probe, FM 4-64 does not fluoresce much in water but fluoresces strongly after binding to the outer plasma membrane, providing clear and distinguishable plasma membrane staining. The binding is rapid and reversible. In this protocol vacuoles in yeast cells are stained with the FM4-64 dye, permitting the use of live-cell imaging if needed.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] Yeast Vacuole Staining with FM4-64
[Bio101] 酵母液泡的FM4-64染色

微生物学 > 微生物细胞生物学 > 细胞染色
作者: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
1/5/2011, 14537 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.18

[Abstract] The lipophilic probe, FM 4-64 does not fluoresce much in water but fluoresces strongly after binding to the outer plasma membrane, providing clear and distinguishable plasma membrane staining. The binding is rapid and reversible. In this protocol vacuoles in yeast cells are stained with the FM4-64 dye, permitting the use of live-cell imaging if needed.

[Abstract] 用FM4-64染料为酵母活细胞液泡染色。

Materials and Reagents

  1. FM4-64 (Life Technologies, Molecular Probes, catalog number: F34653 )
  2. DMSO (Sigma-Aldrich)
  3. YES medium
  4. FM4-64 stock solution
  5. EMM
  6. PBS

Equipment

  1. Water bath
  2. Bench-top centrifuge

Procedure

  1. Grow yeast cells to exponential phase in YES medium at 30 °C. 
  2. Spin down cells and resuspend cell pellet in 500 μl YES + 0.5 μl FM4-64 stock solution (8 mM), so the final concentration of FM4-64 is 8 μM. Keep the cells in the dark (i.e., wrapped in aluminum foil). Incubate cells in a 30 °C water bath for 30 min.
    *FM4-64 stock solution = 8 mM (5 μg/μl) in DMSO (stored at -20 °C).
    *FM4-64 does not efficiently label cells in minimal medium, so even if you grew the cells in EMM in order to maintain a plasmid, you must label cells with FM in YES.
  3. Spin down cells and wash pellet by resuspending in YES to remove free FM4-64. Spin down again and resuspend in 1 ml YES. Transfer cells to a culture tube and add 4 ml YES and then shake at 30 °C for 90 min.
  4. Transfer cells (5 ml) to centrifuge tube and spin 5 min at RT.
  5. Resuspend cell pellet in EMM or PBS.
    EMM does not exhibit nearly as much autofluorescence as does YES, so even if you grew the cells in YES, resuspend the cells in EMM at this step. 
  6. Spot cells on a glass slide and cover with coverslip.
  7. Observe fluorescence in microscope using RFP filter.

Notes

This protocol is a pulse-chase procedure designed to label only the vacuole membranes of yeast cells with FM4-64. You may, however, label the membranes of compartments in the entire endocytic pathway (plasma membrane, early and late endosomal membranes, and vacuole membranes) if you continuously pulse the cells with FM4-64 for 60-120 min (i.e., do not chase in label-free medium). Conversely, you may label only the plasma membrane if you add FM4-64 to cells on ice (of course, you will need to maintain the sample at 0 °C during microscopy, which could prove difficult).

Acknowledgments

This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine. Funding to support different projects that have used this protocol has come from NIH – National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, the Pancreatic Cancer Action Network, and the American Heart Association.

References

  1. Rieder, S. E., Banta, L. M., Kohrer, K., McCaffery, J. M. and Emr, S. D. (1996). Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant. Mol Biol Cell 7(6): 985-999.
  2. Vida, T. A. and Emr, S. D. (1995). A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128(5): 779-792.

材料与试剂

1.     FM4-64 (Sigma)

2.     DMSO (Sigma)

 

实验步骤

1.    30,酵母细胞在YES培养基生长至指数生长阶段。

2.    低速离心沉淀细胞,用500 ul YES+ 0.5 ul FM4-64 母液 (8mM)(终浓度为8 uM)重悬细胞。避光保存,30水浴30分钟。

*FM4-64 母液 = 8mM (5ug/ul) 溶于 DMSO ( -20°C保存)

*FM4-64 在少量培养基中不能有效的标记细胞, 因此,即使是为了质粒数量而使细胞生长在EMM中,也应该在YES培养基上用FM染色。

3.    低速离心沉淀细胞,用YES漂洗、重悬细胞,去除多于的FM4-64。再次沉淀细胞,用1ml YES重悬。将细胞转移至细胞培养管,加入4ml YES30,摇床上孵育90分钟。

4.    转移细胞至离心管,室温离心5分钟。

5.    EMMPBS重悬细胞

EMM的自发荧光与YES并不相同,所以即使细胞在YES培养基中生长,也可用EMM重悬细胞。

6.    将细胞点在载玻片上,盖上盖玻片。

7.    RFP滤镜在显微镜下观察荧光。

注意:以上实验步骤是在脉冲追踪实验中,用FM4-64浸染酵母液泡膜的实验。如果持续用FM4-64脉冲细胞,也可以通过胞吞作用侵染多种膜组分(原生质膜,早期和后期的内质网膜, 液泡膜)相反地,如果只在冰上向细胞中加入FM4-64,那么就只能侵染质膜。(显微镜观察期间也应使样品处于0

 

参考文献

1.        Vida T.A., Emr S.D. (1995). A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. Journal of Cell Biology 128(5): 779-92. 

2.        Rieder S.E., Banta L.M., Kohrer K., McCaffery J.M., Emr S.D. (1996). Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant. Molecular Biology of the Cell 7(6): 985-99. 

 

 

English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Tong, Z. (2011). Yeast Vacuole Staining with FM4-64. Bio-protocol Bio101: e18. DOI: 10.21769/BioProtoc.18; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
9/8/2014 2:11:22 PM  

Henry Elix
University of Pretoria

What is your recipe for the YES medium?

9/24/2014 2:12:20 PM  

Zongtian Tong (Author)
Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, USA


Hi Henry, you can find the recipe of the YES medium at https://www.atcc.org/~/media/3907D6168ED64037BCF4693AE75FBA2C.ashx
I also copied it below:

ATCC medium: 2064 YES Medium
Yeast extract................5.0 g
Glucose.....................30.0 g
L-Adenine..................225.0 mg
L-Histidine................225.0 mg
L-Leucine..................225.0 mg
L-Lysine HCl...............225.0 mg
Uracil.....................225.0 mg
Distilled water..............1.0
Sterilize medium by 0.2 micrometer filtration.
For solid medium:
Add 20.0 g agar to 500 ml distilled water and heat to boiling with
stirring to dissolve. Autoclave agar solution at 121C for 15 minutes.
Cool to 50C. Dissolve nutrient components in 500 ml distilled water and
filter-sterilize. Warm the nutrient solution to 50C. Aseptically combine
and mix the agar and nutrient solutions and dispense as required.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register