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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.

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[Bio101] How to Use an Avestin Emulsiflex C3 Homogenizer to Disrupt Cells

Cell Biology > Cell viability > Cell lysis
Author: 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, 10720 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.11

[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.


How to cite: Tong, Z. (2011). How to Use an Avestin Emulsiflex C3 Homogenizer to Disrupt Cells. Bio-protocol Bio101: e11. DOI: 10.21769/BioProtoc.11; Full Text



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8/17/2011 8:30:49 AM  

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 10:08:41 PM  

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

Hello Mathew,

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

Zongtian

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