Analysis of Immunolabeled Cells by Atomic Force Microscopy, Optical Microscopy, and Flow Cytometry

Neagu, C.R.; Werf, Kees O. van der; Putman, Constant A.J.; Kraan, Yvonne M.; Grooth, B.G. de; Hulst, Niek F. van; Greve, Jan

doi:10.1006/jsbi.1994.1004

Cited by 36 publications

(18 citation statements)

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“…To amplify the gold signal, thin films were treated for 5 min with a silver enhancement kit, which would increase the gold particle size from 30 to 100 nm, depending on treatment time (LI Silver Enhancing Kit, Invitrogen). We chose to use the shortest treatment time possible as large variability in particle size has been shown to increase with time [38]. The samples were then washed with ultra-pure water and air dried in a desiccator overnight prior to AFM imaging.…”

Section: Afm Analysis Of Biointerfaces On Thin Filmsmentioning

confidence: 99%

Oriented, Multimeric Biointerfaces of the L1 Cell Adhesion Molecule: An Approach to Enhance Neuronal and Neural Stem Cell Functions on 2-D and 3-D Polymer Substrates

et al. 2012

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This article focuses on elucidating the key presentation features of neurotrophic ligands at polymer interfaces. Different biointerfacial configurations of the human neural cell adhesion molecule L1 were established on two-dimensional films and three-dimensional fibrous scaffolds of synthetic tyrosine-derived polycarbonate polymers and probed for surface concentrations, microscale organization, and effects on cultured primary neurons and neural stem cells. Underlying polymer substrates were modified with varying combinations of protein A and poly-d-lysine to modulate the immobilization and presentation of the Fc fusion fragment of the extracellular domain of L1 (L1-Fc). When presented as an oriented and multimeric configuration from protein A-pretreated polymers, L1-Fc significantly increased neurite outgrowth of rodent spinal cord neurons and cerebellar neurons as early as 24 h compared to the traditional presentation via adsorption onto surfaces treated with poly-d-lysine. Cultures of human neural progenitor cells screened on the L1-Fc/polymer biointerfaces showed significantly enhanced neuronal differentiation and neuritogenesis on all protein A oriented substrates. Notably, the highest degree of βIII-tubulin expression for cells in 3-D fibrous scaffolds were observed in protein A oriented substrates with PDL pretreatment, suggesting combined effects of cell attachment to polycationic charged substrates with subcellular topography along with L1-mediated adhesion mediating neuronal differentiation. Together, these findings highlight the promise of displays of multimeric neural adhesion ligands via biointerfacially engineered substrates to “cooperatively” enhance neuronal phenotypes on polymers of relevance to tissue engineering.

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Section: Afm Analysis Of Biointerfaces On Thin Filmsmentioning

confidence: 99%

Oriented, Multimeric Biointerfaces of the L1 Cell Adhesion Molecule: An Approach to Enhance Neuronal and Neural Stem Cell Functions on 2-D and 3-D Polymer Substrates

et al. 2012

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“…The latter technique provides a tool for investigation of the surface topography at the air-solid or liquid-solid interface, suitable for imaging with high spatial resolution living and fixed cells in their natural environment (27)(28)(29)(30)(31)(32)(33). In this study, we used SFM in parallel with pbFRET and transmission electron microscopy (TEM) to investigate the distribution of major histocompatibility complex (MHC) class I antigens, key molecules in antigen presentation and cell-mediated cytotoxicity.…”

mentioning

confidence: 99%

Structural hierarchy in the clustering of HLA class I molecules in the plasma membrane of human lymphoblastoid cells.

Damjanovich

Vereb

Schaper

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Major histocompatibility complex (MHC) class I antigens in the plasma membranes of human T (HUT-102B2) and B (JY) lymphoma cells were probed by immunochemical reagents using fluorescence, transmission electron, and scanning force microscopies. Fluorescent labels were attached to monoclonal antibodies W6/32 or KE-2 directed against the heavy chain of HLA class I (A, B, C) and L368 or HB28 against the 82-microglobulin light chain. The topological distribution in the nanometer range was studied by photobleaching fluorescence resonance energy transfer (pbFRET) on single cells. A nonrandom codistribution pattern of MHC class I molecules was observed over distances of 2-10 nm. A second, nonrandom, and larger-scale topological organization of the MHC class I antigens was detected by indirect immunogold labeling and imaging by transmission electron microscopy (TEM) and scanning force microscopy (SFM). Although some differences in antigen distribution between the B-and T-cell lines were detected by pbFRET, both cell lines exhibited similar clustering patterns by TEM and SFM. Such defined molecular distributions on the surfaces of cells of the immune system may reflect an underlying specialization of membrane lipid domains and fulfill important functional roles in cell-cell contacts and signal transduction.The plasma membrane of lymphocytes accommodates many transmembrane proteins, receptors, and antigens, which have a limited mobility and/or occur in oligomeric assemblies (1). Multisubunit structures of key receptors involved in lymphocyte activation, like those of the T-cell receptor-CD3 and the interleukin 2 (IL-2) receptor systems, have been observed (2-5). It is likely that clustering of receptors in the membrane, either preexisting or induced by specific ligands, is important not only in transmembrane signaling but also in antigen presentation and cell-cell communication and contact. Seemingly unrelated integral membrane proteins exhibit a nonrandom pattern of codistribution, as detected by flow cytometric fluorescence energy transfer (FCET), microscope-based photobleaching resonance energy transfer (pbFRET), and lateral mobility measurements or biochemical crosslinking experiments (2,(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Biochemical crosslinking and quantitative FRET can probe the lateral distribution pattern of cell surface antigens and receptors over distances of 2-10 nm (20)(21)(22)(23)(24)(25)(26). Recently, a model based on normalized FCET measurements was proposed for the two-dimensional lateral organization of the intercellular adhesion molecule 1 (ICAM-1) molecule, the IL-2 receptor, and the class I and class II human leukocyte antigen (HLA) molecules (2).The long-range lateral distribution of labeled antigens in the plasma membrane can be detected by electron microscopy and scanning force microscopy (SFM). The latter technique provides a tool for investigation of the surface topography at the air-solid or liquid-solid interface, suitable for imaging with high spatial resolution livi...

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“…Several reports have already described the development and use of such combined platforms. Identification of fluorescence markers on cell membranes or measuring adhesion forces constitute the main applications [15-17]. Other dedicated set-ups have probed their utility in combining fluorescence spectroscopy and SFM for identifying DNA and polystyrene beads [18], lipid organization [19] or light harvesting complexes in membranes [20].…”

Section: Introductionmentioning

confidence: 99%

Molecular recognition of DNA–protein complexes: A straightforward method combining scanning force and fluorescence microscopy

2010

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Combining scanning force and fluorescent microscopy allows simultaneous identification of labeled biomolecules and analysis of their nm level architectural arrangement. Fluorescent polystyrene nano-spheres were used as reliable objects for alignment of optical and topographic images. This allowed the precise localization of different fluorescence particles within complex molecular assemblies whose structure was mapped in nanometer detail topography. Our experiments reveal the versatility of this method for analysis of proteins and protein-DNA complexes.

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Analysis of Immunolabeled Cells by Atomic Force Microscopy, Optical Microscopy, and Flow Cytometry

Cited by 36 publications

References 0 publications

Oriented, Multimeric Biointerfaces of the L1 Cell Adhesion Molecule: An Approach to Enhance Neuronal and Neural Stem Cell Functions on 2-D and 3-D Polymer Substrates

Oriented, Multimeric Biointerfaces of the L1 Cell Adhesion Molecule: An Approach to Enhance Neuronal and Neural Stem Cell Functions on 2-D and 3-D Polymer Substrates

Structural hierarchy in the clustering of HLA class I molecules in the plasma membrane of human lymphoblastoid cells.

Molecular recognition of DNA–protein complexes: A straightforward method combining scanning force and fluorescence microscopy

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