Combined Intracellular and Surface Staining: Immunofluorescence of Cytokines in T Cells

  • Mario Assenmacher
Part of the Springer Lab Manual book series (SLM)


Detection of intracellular proteins by immunofluorescence allows to determine frequency, light scatter- and surface-immunophenotype of protein producing cells, irrespective of whether the protein is to be secreted, membrane-bound or localized in the cytoplasm. For cytoplasmatic or nuclear proteins this is the only way to analyse them by flow cytometry. Unfortunately, to date no method is known for cytoplasmatic immunofluorescence of live cells. But recently immunofluorescence of live cells according to secreted products, including cytokines, became possible with the cellular affinity matrix technology (Manz 1995, Assenmacher 1998, see → Kap. 6). In order to allow staining antibodies to penetrate the cell membrane, cells have to be fixed and the membranes permeabilized. The choice of the appropriate fixation method depends on the protein and its intracellular location, and on the further use of the cells to be analysed. For several applications fixation in formaldehyde and permeabilization of cell membranes by saponin (Willingham 1985) has been used successfully, including assessment of cytokines (Sander 1991; Jung 1993; Schmitz 1993; Assenmacher 1994). Formaldehyde is a crosslinking fixative with good preservation of cell morphology. The plant glycoside saponin, a mild nonionic detergent, complexes with membrane cholesterol and other unconjugated β-hydroxysterols leading to ring-shaped complexes with a central pore of about 8nm in diameter (Bangham 1962; Glauert 1962). These pores allow passage of molecules up to several hundred kD large. Since saponin acts in a reversible way it has to be present in all incubation and washing steps. Many nowadays commercially available fixation and permeabilisation buffers contain formaldehyde and saponin, respectively.


Tetanus Toxoid Surface Staining Mouse Spleen Cell Mild Nonionic Detergent Anti Cytokine 
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© Springer-Verlag Berlin Heidelberg 2000

Authors and Affiliations

  • Mario Assenmacher
    • 1
  1. 1.Miltenyi Biotec GmbHBergisch GladbachGermany

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