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

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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.

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.

简介

亲脂性探针FM 4-64在水中不发荧光,但在与外部质膜结合后发出强烈荧光,提供清晰和可区分的质膜染色。 结合是快速和可逆的。 在该协议中,酵母细胞中的液泡用FM4-64染料染色,如果需要,允许使用活细胞成像。

材料和试剂

  1. FM4-64(Life Technologies,Molecular Probes,目录号:F34653)
  2. DMSO(Sigma-Aldrich)
  3. YES培养基
  4. FM4-64储备液
  5. EMM
  6. PBS

设备

  1. 水浴
  2. 台式离心机

程序

  1. 在30℃下在YES培养基中将酵母细胞培养至指数期。
  2. 旋转细胞并重悬细胞沉淀在500μlYES +0.5μlFM4-64储备溶液(8mM)中,因此FM4-64的最终浓度为8μM。保持细胞在黑暗中(,即,包裹在铝箔)。将细胞在30℃水浴中孵育30分钟 * FM4-64储备溶液= 8mM(5μg/μl)在DMSO中(储存在-20℃) * FM4-64不能有效地标记基本培养基中的细胞,因此即使您在EMM中生长细胞以维持质粒,您必须在YES中标记具有FM的细胞。
  3. 旋转细胞和洗涤沉淀通过重悬在YES,以删除游离FM4-64。再次旋转并重悬在1毫升是。转移细胞到培养管,并加入4毫升,然后在30℃摇动90分钟。
  4. 转移细胞(5ml)离心管,在室温下旋转5分钟。
  5. 在EMM或PBS中重悬细胞沉淀 EMM没有表现出与YES相同的自发荧光,所以即使你在YES中生长细胞,在这一步骤重新悬浮细胞在EMM中。
  6. 在玻璃幻灯片的斑点细胞和盖子与盖片。
  7. 使用RFP滤光片观察显微镜中的荧光

笔记

该方案是设计为仅用FM4-64标记酵母细胞的液泡膜的脉冲追踪程序。 然而,如果你连续脉冲细胞与FM4-64 60-120分钟(即,不追逐),你可以标记整个内吞途径(细胞膜,早期和晚期endosomal膜和液泡膜)隔室的膜 在无标记培养基中)。 相反,如果你将FM4-64添加到冰上的细胞,你可以只标记质膜(当然,在显微镜下你需要将样品保持在0°C,这可能很难)。

致谢

该协议已经在约翰霍普金斯医学院的Espenshade实验室中修改和改编。 资助支持不同的项目,使用这个协议来自NIH - 国家心脏,肺和血液研究所,国家过敏和传染病研究所,胰腺癌行动网络和美国心脏协会。

参考文献

  1. Rieder,S.E.,Banta,L.M.,Kohrer,K.,McCaffery,J.M.and Emr,S.D。(1996)。 多层内体样间室积聚在酵母vps28液泡蛋白分选突变体中。 Mol Biol Cell 7(6):985-999。
  2. Vida,T.A。和Emr,S.D。(1995)。 用于在酵母中显现液泡膜动力学和内吞作用的新的活性染色。 J Cell Biol 128(5):779-792
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tong, Z. (2011). Yeast Vacuole Staining with FM4-64. Bio-protocol Bio101: e18. DOI: 10.21769/BioProtoc.18;
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Henry Elix
University of Pretoria
What is your recipe for the YES medium?
9/8/2014 2:11:22 PM Reply
Zongtian Tong
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.

9/24/2014 2:12:20 PM