Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer.

Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer. Research Abstract Details 

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Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer. Abstract Text:

Paul V Beum,Margaret A Lindorfer,Brian E Hall,Thaddeus C George,Keith Frost,Philip J Morrissey,Ronald P Taylor,

Binding of the chimeric, humanized anti-CD20 mAb Rituximab (RTX) to B lymphocytes activates complement and promotes covalent deposition of C3 fragments (C3b/iC3b) on cells. Previous fluorescence microscopy studies, based on examination of B cell lines and of blood samples from RTX-treated CLL patients, suggest that C3b/iC3b is closely associated with cell-bound RTX. We examined Raji cells opsonized with serum and RTX with the ImageStream imaging flow cytometer. Cells were stained with fluorescently-labeled RTX and mAbs specific for C3b/iC3b fragments or for human IgG, and then imaged using the ImageStream cytometer and analyzed with an algorithm (Similarity Bright Detail Score, SBDS) which tests for co-localization of fluorescent probes. SBDS, calculated on 10,000 cells, verified that the majority of deposited C3b/iC3b is co-localized with bound RTX. In contrast, when cells were first opsonized in serum alone, washed and then reacted with RTX, SBDS confirmed that RTX and C3b/iC3b are poorly co-localized, thus demonstrating that cell-bound RTX directs deposition of C3b. In addition, a sulfhydryl-specific probe, maleimide conjugated to AF488, exhibited substantial co-localization with an anti-C3b/iC3b mAb on Raji cells opsonized with RTX and serum, thus validating maleimide labeling as an alternative for detecting cell-bound C3b/iC3b. The digital imaging method described should have wide applicability for quantitative analysis of co-localization.

Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer. Publishing Authors By Initials

PV Beum,MA Lindorfer,BE Hall,TC George,K Frost,PJ Morrissey,RP Taylor,

For similar proteins research abstracts see: proteins research

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Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer. Journal Published:

PUBLICATION TYPE: Research Support, N.I.H., Extr

Journal: Journal of immunological methods

VOLUME: 317

Page Numbers: 90-9

Journal Abbreviation: J. Immunol. Methods

ISSN: 0022-1759

DAY: 10

MONTH: 10

YEAR: 2006

Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 1305440

Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer. Keywords Mesh Terms:

KEYWORDS: Proteins

MESH TERMS: analysis

Chemical & Substance for Abstract: Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer. Information

Substance Name: Complement System Proteins

Registry Number: 9007-36-7

Grant and Affiliation Information for Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer.

AFFILIATION: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

Country: Netherlands

AGENCY: United States NCI

GRANT: 9 R44 CA01798-02

ACRONYM: CA

MEDLINETA: J Immunol Methods

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