Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer.
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.
Beum
Paul V
PV
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Lindorfer
Margaret A
MA
Hall
Brian E
BE
George
Thaddeus C
TC
Frost
Keith
K
Morrissey
Philip J
PJ
Taylor
Ronald P
RP
D000465:Algorithms; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D058846:Antibodies, Monoclonal, Murine-Derived; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D003165:Complement System Proteins / Q000378:metabolism*; D003952:Diagnostic Imaging; D005434:Flow Cytometry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011506:Proteins / Q000032:analysis; D000069283:Rituximab
D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D000970:Antineoplastic Agents; D011506:Proteins; D000069283:Rituximab; D003165:Complement System Proteins