Permeabilisation

Cell source Stimulation Secretion stop Fixation Permeabilisation Staining Protocol

 

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To detect intracellular you have to make it possible for your antibody (Ab) or ligand to enter the cell. There are many different way s to do that. First of all you have to stabilize your cell by fixation!
After fixation you got a wide choice of different chemicals that make it able for your Ab or ligand to enter the cell. There are already companies that sell buffers that not only fix but also permeabilize your sample at the same time. When you would use acetone for fixation of cyto-spins these cells will also be fixated and permeabilized in one go.
For flow cytometry we often use formaldehyde to fixate and than we need a second chemical to permeabilize the cells. Saponin is one of those chemicals and used most often. But also Triton X-100 and n-octyl-D-glucopyranoside are chemicals you can use to permeabilize the sample. 
Some conjugates might be too big to enter cells. You have to think of the tandem dyes like PE-Cy5 than.

Saponin.

Saponin is isolated from Quillaja bark. It is a glucoside with a high affinity for cholesterol. It will replace the cholesterol in the membrane and make pores big enough to have Ab's enter the cell (Willingham, et al.). Since the intercalation is a reversible process it is important to have saponin present in all your incubation and wash steps. Minimum concentration is 0.1%. Normally used in concentration between 0.2 and 0.4%. This mainly depending on the batch since the purity of the isolation is not always as good. The browner the preparation the higher the the concentration has to be.

Triton.

This widely used non-ionic surfactant is a detergent which permeabilizes the cell membrane. It is often used to isolated membrane components under non-denaturizing conditions. Just as for Saponin hold true; too high concentrations simply will dissolve the cells.

n-octyl-D-glucopyranoside.

n-octyl-D-glucopyranoside or n-Octyl glucoside has the same characteristics as Triton. This chemical is also an non-ionic detergent used for the solubilization and isolation of membrane proteins.

 

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 Suggestions, literature and protocols are welcome at icstaining@schuitemaker.info. Copyright 2000 - 2005.
Page was last updated: 05-03-2005