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  A microassay for the pore-forming activity of complement, perforin, and other cytolytic proteins based on confocal laser scanning microscopy

Sauer, H., Pratsch, L., & Peters, R. (1991). A microassay for the pore-forming activity of complement, perforin, and other cytolytic proteins based on confocal laser scanning microscopy. Analytical Biochemistry, 194(2), 418-424. doi:10.1016/0003-2697(91)90251-n.

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 Creators:
Sauer, Heinrich1, Author              
Pratsch, Lothar2, Author              
Peters, Reiner1, Author              
Affiliations:
1Department of Cell Physiology, Max Planck Institute of Biophysics, Max Planck Society, ou_3264817              
2Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              

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 Abstract: A fluorescence microscopic assay for the activity of complement, perforin, and other cytolytic proteins which form transmembrane pores in cellular membranes is described. The assay was worked out and tested with red blood cell membranes (ghosts) and was then applied to intact hemoglobin-free cells. Resealed human erythrocyte ghosts were incubated with complement or perforin. A small polar fluorescent probe (fluorescein-labeled 1-kDa dextran, FD1) which permeates through complement and perforin pores but not through normal cell membranes was added to the samples. The capability of the confocal laser scanning microscope (CLSM) to generate thin optical sections was exploited to visualize and quantitate fluorescence inside single ghosts and thus determine the fraction of ghosts which had become permeable for FD1. The activity of complement or perforin was quantitated by plotting the fraction of permeable cells versus the concentration of the pore-forming protein. The results were in good agreement with those of a conventional hemolytic assay. The CLSM-based assay was then applied to intact hemoglobin-free cells for which only few alternative assays are available. Compared to conventional hemolytic assays for the activity of pore-forming proteins the assay described here can be applied to a large variety of natural and artificial membrane systems. The assay can be performed under nonlysing conditions. Furthermore, the assay is simple, relatively fast, and requires only extremely small amounts of cells and pore-forming proteins.

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Language(s): eng - English
 Dates: 1990-10-092004-12-061991-05-01
 Publication Status: Published in print
 Pages: 7
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/0003-2697(91)90251-n
PMID: 1862943
 Degree: -

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Title: Analytical Biochemistry
Source Genre: Journal
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Publ. Info: San Diego, CA : Academic Press
Pages: - Volume / Issue: 194 (2) Sequence Number: - Start / End Page: 418 - 424 Identifier: ISSN: 0003-2697
CoNE: https://pure.mpg.de/cone/journals/resource/954922644003