Detergents are used in a broad range of scientific procedures but they often seem to be used interchangeably and figuring out why one is used over another in different applications can be quite confusing. In this post we’re going to concentrate on the use of detergents for permeabilisation of cell membranes in intracellular staining.

If you’re unsure as to how detergents work you might find our previous post on the topic useful, otherwise sit tight and let’s see if we can help clear up a few things.

What method or detergent should I choose?
There are many ways in which intracellular staining can be achieved but the choice of which method and reagents to use should be carefully selected based on your required target and the method of assessment.

For instance, if your assay will use flow-cytometry it is usually important to preserve the morphology and light scattering characteristics of the cell. The choice of fixation and level of permeabilisation can drastically impact on a cell’s morphology. Equally, what type of molecule your target is and where it resides can be important. If it is a membrane protein, or if you are using cell surface labelling (such as a CD marker) in addition to intracellular staining to identify your cell type, you may not want to use a detergent that permeabilises membranes by removing membrane proteins.

Commonly cell membrane permeabilisation is achieved by using either:

A. Organic solvents (i.e. methanol/acetone), which both fix and permeabilise membranes by dissolving membrane lipids and coagulating proteins.
B. Detergents; these just permeabilise cell membranes by creating pores. The most frequently used being (Saponin, Trition-X-100 or Tween-20).

How do the individual detergents work?

  • Saponin- selectively interacts with membrane cholesterols, forming pores and/or removing cholesterols from the membrane[1].
  • Triton-X and Tween-20 are non-selective and interact with both protein and lipids creating pores and/or removing them from the membrane [2].

For further reading and a list of additional detergents see [3]

Advantages & disadvantages of different detergents

  • Reversible- Saponins will wash out so following permeabilisation if further downstream steps are required wash buffers will also need to contain saponin.
  • Will not permeabilise the nuclear membrane.
  • Can be used to selectively permeabilise mammalian cell membranes based on their cholesterol content and concentration of saponin.
  • Maintains the integrity of protein surface antigens

Triton-X-100 & Tween-20
In the literature for the purpose of permeabilisation there appears to be very little difference between the non-ionic detergents Triton-X and Tween-20. This difference may be greater when used in other applications. However, for this post we have grouped them together.

  • Non-selective- may extract both proteins and lipids.
  • Permeabilise all lipid bilayers
  • High concentrations or long incubation times are likely to lyse cells.
  • Both absorb UV light because they contain a phenyl ring. This could have implications on your read out if you use stains that overlap with this.
  • If your target is a lectin these detergents are much less likely to impact your results than saponin.

So there you have it! There are a few decisions to be made and should be weighed against what you’re looking for and how you’re going to measure your results. However, I do feel there is a common theme….

  • If you’re using microscopy and cells fixed to a glass slide you can try the quick and dirty approach of fix/perm with an organic solvent.
  • If you’re using flow-cytometry, using cell surface stains along with intracellular stains saponin is the common option.
  • However, if you’re using flow-cytometry and your cellular target is in the nucleus, the detergent of choice would be Tween-20/Triton-X [4].

What do you think? Please let us know by joining in the discussion either here or on our twitter feed.

1. Lacaille-Dubois MA1, Wagner H. A review of the biological and pharmacological activities of saponins. Phytomedicine. 1996 Mar;2(4):363-86. doi: 10.1016/S0944-7113(96)80081-X. PMID : 23194774
2 Permeabilization of cell membranes. Methods Mol Biol. 2010;588:63-6. doi: 10.1007/978-1-59745-324-0_9.

4. Amidzadeh Z, Behbahani AB, Erfani N, et al. Assessment of Different Permeabilization Methods of Minimizing Damage to the Adherent Cells for Detection of Intracellular RNA by Flow Cytometry. Avicenna Journal of Medical Biotechnology. 2014;6(1):38-46. PMID:24523954

– From Liz and the CiteAb team