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[Bio101] How to Use an Avestin Emulsiflex C3 Homogenizer to Disrupt Cells
[Bio101] 如何使用Avestin公司Emulsiflex C3型高压均质机进行细胞破碎

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Abstract

The EmulsiFlex-C3 homogenizer is powered by an electric motor. The pump does not require a compressor for it to run. This equipment can be used to disrupt cells at a large scale. The EmulsiFlex-C3 has a fixed flow-through capacity of 3 L/h. It has the ability to process samples as small as 10 ml. The homogenizing pressure is adjustable between 500 and 30,000 psi. In this protocol, we describe the use of the Avestin Emulsiflex C3 Homogenizer to disrupt S. pombe and S. cerevisiae cells.

Materials and Reagents

  1. S. pombe cells
  2. S. cerevisiae cells
  3. DI water

Equipment

  1. Avestin Emulsiflex C3 homogenizer (Avestin®)

    Figure 1. Avestin Emulsiflex C3 homogenizer

  2. Standard laboratory bench-top light microscope

Procedure

  1. Switch on the homogenizer.
  2. Turn on nitrogen. Pressure reads 80 psi.
  3. Unscrew the funnel cap. Check if the funnel cap is on to make sure ethanol does not evaporate.
  4. Turn red stop knob clockwise and push green knob to start.
  5. Pump residual ethanol out of the tubing. 
  6. Pour DI water into the funnel to wash ethanol out. Keep air pressure on occasionally to make sure no cell debris is left from the last user.
  7. Before loading samples, take the funnel off and roll it on ice to keep it cool. Install the funnel back to the top. 
  8. Put the steel coil heat exchanger into ice to cool down the samples.
  9. Load your samples into the funnel. Turn on the homogenizer. Let the samples run through the tubing back to the funnel before air pressure is on.    
  10. Turn on air pressure. Air pressure at 40 psi, gauge pressure ≥ 20,000 psi and <25,000 psi. The maximum pressure is 30,000 psi. Leave the tubing in a sample collection tube chilled on ice.
  11. S. pombe samples need to be passed through 5~6 times to reach 80~90% efficiency. S. cerevisiae samples need to be passed through 8~9 times to reach 80~90% lysis efficiency. 
  12. Check samples under a standard light microscope.
  13. If the homogenizer is clogged by the samples, cap the funnel and blow with nitrogen tube.
  14. After samples are done, take off the funnel and rinse it with DI water.
  15. Run more water to flush out cell debris. Keep the air pressure on occasionally.
  16. Run ethanol and leave 1/3 of a funnel volume of ethanol in the funnel.

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. Breslow, D. K., Collins, S. R., Bodenmiller, B., Aebersold, R., Simons, K., Shevchenko, A., Ejsing, C. S. and Weissman, J. S. (2010). Orm family proteins mediate sphingolipid homeostasis. Nature 463(7284): 1048-1053.

简介

EmulsiFlex-C3匀浆器由电动机提供动力。 该泵不需要压缩机来运行。 该设备可用于大规模破坏细胞。 EmulsiFlex-C3具有3 L/h的固定流通能力。 它具有处理小至10ml的样品的能力。 均化压力可在500和30,000psi之间调节。 在这个协议,我们描述使用Avestin Emulsiflex C3均质器破坏。 pombe 和 S。 酿酒酵母细胞。

材料和试剂

  1. pombe 细胞
  2. 酿酒酵母细胞
  3. 去离子水

设备

  1. Avestin Emulsiflex C3匀浆器(Avestin )

    图1. Avestin Emulsiflex C3匀浆器

  2. 标准实验室台式光学显微镜

程序

  1. 打开均化器。
  2. 打开氮气。 压力读数为80 psi。
  3. 拧下漏斗盖。 检查漏斗盖是否打开,以确保乙醇不会蒸发。
  4. 顺时针转动红色停止旋钮,并按绿色旋钮开始。
  5. 将残余乙醇泵出管道。
  6. 将去离子水倒入漏斗中以洗涤乙醇。 偶尔保持空气压力,以确保没有细胞碎片留在最后一个用户。
  7. 在装样前,取下漏斗,在冰上滚动,保持冷却。 将渠道安装回顶部。
  8. 将钢卷热交换器放入冰中冷却样品。
  9. 将样品装入漏斗。 打开均化器。 让样品在空气压力开启前通过管道返回漏斗。    
  10. 打开气压。 空气压力为40psi,表压为≥20,000psi且小于25,000psi。 最大压力为30,000psi。 将管放置在冰上冷冻的样品收集管中。
  11. pombe 样品需要通过5〜6次,达到80〜90%的效率。

    酿酒酵母样品需要通过8〜9次以达到80〜90%的裂解效率。
  12. 在标准光学显微镜下检查样品。
  13. 如果均质器被样品阻塞,盖上漏斗并用氮气管吹扫。
  14. 样品完成后,取出漏斗,用去离子水冲洗。
  15. 运行更多的水冲洗细胞碎片。 偶尔保持气压。
  16. 运行乙醇,在漏斗中留下漏斗中1/3体积的乙醇

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. Breslow,D.K.,Collins,S.R.,Bodenmiller,B.,Aebersold,R.,Simons,K.,Shevchenko,A.,Ejsing,C.S。和Weissman,J.S。(2010)。 Orm 家族蛋白介导鞘脂稳态。 自然 463(7284):1048-1053。
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引用:Tong, Z. (2011). How to Use an Avestin Emulsiflex C3 Homogenizer to Disrupt Cells. Bio-protocol Bio101: e11. DOI: 10.21769/BioProtoc.11;
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Matthew
Univesity of Colorado, Colroado Springs
Hi,

My name is Matthew and may lab recently purchased a homogenizer.

I was wondering why you used nitrogen?

The manual suggests using compressed air over nitrogen, although it states that using compressed nitrogen was fine.

Will it make any difference?

Thanks.

mpeetz@uccs.edu
8/17/2011 8:30:49 AM Reply
Zongtian Tong
Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, USA

Hello Mathew,

It shouldn't make a difference with compressed air or nitrogen. However, consult with the manufacturer first.

Zongtian

8/17/2011 10:08:41 PM