scholarly journals Evidence for membrane differentiation in polarised leucocytes: the distribution of surface antigens analysed with Ig-gold labelling

1990 ◽  
Vol 95 (3) ◽  
pp. 471-479
Author(s):  
W.S. Haston ◽  
A.F. Maggs
Keyword(s):  
Random Distribution ◽  
Mononuclear Cells ◽  
Surface Antigens ◽  
Membrane Flow ◽  
Thin Sections ◽  
Gold Particles ◽  
Hla Dr ◽  
Transmission Electron ◽  
Morphological Polarity ◽  
Mononuclear Leucocytes

The distribution of a number of leucocyte surface antigens was studied on both round and polarised neutrophil or mononuclear leucocytes using Ig-gold conjugates with transmission electron microscopy. Thin sections of cells, which had been lightly fixed before antibody labelling, were analysed using a statistical method to determine: (1) whether the antigens had a non-random distribution or ‘clustering’ over the cell surface; and (2) whether there was any overall bias in labelling to give a polarised distribution. Comparison between the results of this analysis and cell morphology were made. The results indicated that with the antigens investigated here, CD45, CD15, HLA-DR and CR3, the majority of polarised cells had a calculated direction of overall asymmetry of gold particles that was aligned with the long axis of morphological polarity. Maximal asymmetry was seen in polarised cells labelled for CD45 and HLA-DR, with labelling ratios of up to 6:1 between the front and back of the cell. A number of round mononuclear cells demonstrated significant polarisation of gold particles but this had no apparent morphological correlation and, in general, round cells showed a low degree of asymmetry. However, there was evidence that both round and polarised cells had a non-random distribution or ‘clustering’ of gold particles, which was more marked in morphologically polarised cells and particularly significant in polarised neutrophil leucocytes labelled for CR3. The significance of these results for models of cell locomotion involving membrane flow is discussed.

Quantification of cell-surface expressed antigenic sites with 5-nm gold markers: Getting close to biologically relevant data

1989 ◽  
Vol 47 ◽  
pp. 1050-1051
Author(s):  
Etienne de Harven ◽  
Hilary Christensen ◽  
Richard Leung ◽  
Cameron Ackerley
Keyword(s):  
Cell Surface ◽  
Human Peripheral Blood ◽  
Contour Length ◽  
Thin Sections ◽  
Gold Particles ◽  
Biologically Relevant ◽  
Transmission Electron ◽  
Entire Cell ◽  
Electron Imaging ◽  
Cell Contour

The T-derived subset of human peripheral blood normal lymphocytes has been selected as a model system to study the usefulness of 5 nm gold markers for quantification of single epitopes expressed on cell surfaces. The chosen epitopes are parts of the CD3 and CD5 molecules and can be specifically identified by hybridoma produced monoclonal antibodies (MoAbs; LEU-4 and LEU-1; Becton-Dick- inson, Mountain view, CA) . An indirect immunolabeling procedure, with goat anti-murine IgG adsorbed on the surface of 5 nm colloidal gold particles (GAM-G5, Janssen Pharmaceutica, Beerse, Belgium) has been used. Backscattered Electron Imaging (BEI) in a field emission scanning electronmicroscope (SEM) and transmission electron microscopy of thin sections of lymphocytes labeled before plastic embedding, were both used to identify and quantitate gold labeled cell surface sites, Estimating that the thickness of “silver” sections is approximately 60 nm and counting the number of gold particles on the entire cell perimeter, we calculated that, for LEU-4, the number of markers per um2 of cell surface is in the 140-160 range (Fig.l). Cell contour length measurements indicated that the surface of one lymphocyte is approximately 130-160 um2 that of a smooth sphere of identical diameter, reflecting the role of microvilli in expanding the surface area. The total number of gold labeled sites on the surface of one lymphocyte averages, therefore between 20,000 and 24,000 per cell.

Antigen-presenting Cells in Human Radicular Granulomas

2008 ◽  
Vol 87 (6) ◽  
pp. 553-557 ◽  
Author(s):  
T. Kaneko ◽  
T. Okiji ◽  
R. Kaneko ◽  
J.E. Nör ◽  
H. Suda
Keyword(s):  
Electron Microscopy ◽  
T Cells ◽  
Dendritic Cells ◽  
Antigen Presenting Cells ◽  
Gold Particles ◽  
Immunological Responses ◽  
Hla Dr ◽  
Transmission Electron ◽  
Antigen Presenting ◽  
Cytoplasmic Processes

Substantial numbers of dendritic cells have been detected in radicular granulomas. To test the hypothesis that local antigen presentation from dendritic cells to T-cells is involved critically in immunological responses within radicular granulomas, we compared characteristics of dendritic cells and macrophages by morphological and biological analyses. Under light microscopy, HLA-DR+ and CD68+ cells showed diverse profiles, including dendritic-shaped cells. Transmission electron microscopy revealed that HLA-DR+ dendritic cells, with long cytoplasmic processes and lacking distinct phagosomes, were concentrated in the lymphocyte-rich area. HLA-DR alpha-chain, CD83, and CD86 mRNAs from HLA-DR+ dendritic cells, and CD28 mRNA from CD28+ T-cells were up-regulated in lymphocyte-rich area. Scanning electron microscopy demonstrated that the density of gold particles on dendritic cells was higher than that on HLA-DR+ macrophages. These results suggest that dendritic cells in radicular granulomas are associated with local defense reactions as stronger antigen-presenting cells, as compared with macrophages.

Characterization of beam-induced thinning and shrinkage of semi-thick sections in the HVEM

1990 ◽  
Vol 48 (3) ◽  
pp. 752-753
Author(s):  
J.R. Kremer ◽  
E.T. O'Toole ◽  
G.P. Wray ◽  
D.M. Mastronarde ◽  
S.J. Mitchell ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Transmission Electron Microscope ◽  
Colloidal Gold ◽  
Similar Data ◽  
Thin Sections ◽  
Gold Particles ◽  
Dose Rates ◽  
Transmission Electron ◽  
Conventional Transmission Electron Microscope

It is well known that irradiation of plastic sections in a conventional transmission electron microscope (cTEM) causes specimen thinning and distortion. Thinning has been observed in the cTEM using several embedding media, using methods such as shrinkage of ordered paracrystalline structures, and shrinkage of sections coated with colloidal gold markers. The total thinning observed in the cTEM (80kev) is 30-50% for thin sections of epon araldite, but similar data do not exist for the HVEM at 1000 kev. Here we describe beam induced thinning and shrinkage of 0.2um sections in the HVEM.Experiments were performed using 0.2um sections of EPOX 812/Araldite or LX112 with 15 nm and 30 nm gold particles affixed to either surface of the section. The sections were initially tilted to approximately 25° and irradiated with known dose rates. Micrographs were taken at different times between 0-20 minutes then the sections were tilted back to 0° for a reference micrograph.

scholarly journals Phenotypic and genetic evaluation of human monocyte-derived dendritic cells generated from whole blood for immunotherapy

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yousri M. Hussein ◽  
Doaa M. Hendawy ◽  
Abdalrahman N. Alghamdy ◽  
Nermin Raafat
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Surface Antigens ◽  
Interleukin 4 ◽  
Blood Monocytes ◽  
Hla Dr ◽  
Significant Difference

Abstract Background Dendritic cells (DCs) recognize different pathogens and cancer cells and activate the adaptive immune response. The generation of effective DC-based cancer vaccines depends on the appropriate differentiation of monocytes in vitro. This study aimed to standardize a protocol for the in vitro differentiation of human peripheral blood monocytes into immature DCs upon treatment with growth factors and generate monocyte-derived DCs (MoDCs). Peripheral blood mononuclear cells were separated from peripheral blood. After monocyte enrichment by plastic adhesion, monocytes were cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 to generate immature DCs. The cells were examined by microscopy. Using flow cytometry, DCs were evaluated for the expression of the CD83 and HLA-DR surface antigens, for the uptake of fluorescein isothiocyanate conjugated dextran, and also for the expression of CD80 and CD86 mRNA. Results CD80 and CD86 genes expression was upregulated at day six and exhibited a significant difference (P < 0.05). DCs showed positive expression of the CD83 and HLA-DR surface antigens by flow cytometry and FITC-conjugated dextran uptake. Conclusion This study represents a preliminary trial to generate immature MoDCs in vitro from blood monocytes collected by the flask adherence method. It offers a panel of surface markers for DCs characterization and provides Immature DCs for experimental procedures after 6 incubation days.

scholarly journals Monoclonal antibodies bound to subunits of the integrin GPIIb-IIIa are internalized and interfere with filopodia formation and platelet aggregation

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1564-1571 ◽  
Author(s):  
WM Isenberg ◽  
DF Bainton ◽  
PJ Newman
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Membrane Flow ◽  
Thin Sections ◽  
Fibrinogen Receptor ◽  
Platelet Morphology ◽  
Fibrinogen Binding ◽  
Transmission Electron ◽  
Cytoskeletal Rearrangements

Abstract The monoclonal antibodies Tab and AP3 are directed, respectively, against GPIIb and GPIIIa, the subunits of the platelet fibrinogen receptor. When added together to platelets, these antibodies prevent adenosine diphosphate (ADP)-induced platelet aggregation, despite normal fibrinogen binding (Newman et al, Blood 69:668, 1987). To explore the cellular requirements of aggregation after fibrinogen binding, we used several techniques to study platelets treated with Tab and AP3, then stimulated with ADP. We used scanning and transmission electron microscopy to evaluate platelet morphology, immunolabel- surface replication to determine whether individual GPIIb-IIIa complexes clustered, immunocytochemistry on frozen thin sections to study the subcellular distribution of the integrin GPIIb-IIIa and fibrinogen, and biochemical methods to assess the activation of the platelet cytoskeleton. We found that the treated cells had short, blunted projections instead of normal filopodia. Other morphologic abnormalities, apparent in thin section, were aberrantly placed alpha- granules and microtubules, and a prominent, worm-like, fibrinogen- filled surface-connected canalicular system. Biochemical analysis suggested that such platelets undergo massive actomyosin-controlled membrane flow, which serves to sequester GPIIb-IIIa and makes the platelets refractory to aggregation. We conclude that aggregation requires the formation of long, slender filopodia, probably directed by cytoskeletal rearrangements after activation, and that the transmembrane GPIIb-IIIa complex may play a role in signaling these events.

scholarly journals Monoclonal antibodies bound to subunits of the integrin GPIIb-IIIa are internalized and interfere with filopodia formation and platelet aggregation

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1564-1571
Author(s):  
WM Isenberg ◽  
DF Bainton ◽  
PJ Newman
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Membrane Flow ◽  
Thin Sections ◽  
Fibrinogen Receptor ◽  
Platelet Morphology ◽  
Fibrinogen Binding ◽  
Transmission Electron ◽  
Cytoskeletal Rearrangements

The monoclonal antibodies Tab and AP3 are directed, respectively, against GPIIb and GPIIIa, the subunits of the platelet fibrinogen receptor. When added together to platelets, these antibodies prevent adenosine diphosphate (ADP)-induced platelet aggregation, despite normal fibrinogen binding (Newman et al, Blood 69:668, 1987). To explore the cellular requirements of aggregation after fibrinogen binding, we used several techniques to study platelets treated with Tab and AP3, then stimulated with ADP. We used scanning and transmission electron microscopy to evaluate platelet morphology, immunolabel- surface replication to determine whether individual GPIIb-IIIa complexes clustered, immunocytochemistry on frozen thin sections to study the subcellular distribution of the integrin GPIIb-IIIa and fibrinogen, and biochemical methods to assess the activation of the platelet cytoskeleton. We found that the treated cells had short, blunted projections instead of normal filopodia. Other morphologic abnormalities, apparent in thin section, were aberrantly placed alpha- granules and microtubules, and a prominent, worm-like, fibrinogen- filled surface-connected canalicular system. Biochemical analysis suggested that such platelets undergo massive actomyosin-controlled membrane flow, which serves to sequester GPIIb-IIIa and makes the platelets refractory to aggregation. We conclude that aggregation requires the formation of long, slender filopodia, probably directed by cytoskeletal rearrangements after activation, and that the transmembrane GPIIb-IIIa complex may play a role in signaling these events.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

Two- or three-way observations of the identical cell elements within the same sections by LM, TEM and/or SEM

1978 ◽  
Vol 36 (2) ◽  
pp. 82-83 ◽  
Author(s):  
Nakazo Watari ◽  
Yasuaki Hotta ◽  
Yoshio Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Fine Structure ◽  
Light Microscopy ◽  
Thin Sections ◽  
Transmission Electron ◽  
Identical Cell ◽  
Electron Microscopes ◽  
Scanning Electron

It is very useful if we can observe the identical cell elements within the same sections by light microscopy (LM), transmission electron microscopy (TEM) and/or scanning electron microscopy (SEM) sequentially, because, the cell fine structure can not be indicated by LM, while the color is; on the other hand, the cell fine structure can be very easily observed by EM, although its color properties may not. However, there is one problem in that LM requires thick sections of over 1 μm, while EM needs very thin sections of under 100 nm. Recently, we have developed a new method to observe the same cell elements within the same plastic sections using both light and transmission (conventional or high-voltage) electron microscopes.In this paper, we have developed two new observation methods for the identical cell elements within the same sections, both plastic-embedded and paraffin-embedded, using light microscopy, transmission electron microscopy and/or scanning electron microscopy (Fig. 1).

SEM Examination of a Used Diamond Knife

1971 ◽  
Vol 29 ◽  
pp. 452-453
Author(s):  
J. Temple Black
Keyword(s):  
Electron Microscope ◽  
High Voltage ◽  
High Magnification ◽  
Metallic Materials ◽  
Wide Spread ◽  
Thin Sections ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Scanning Electron ◽  
Diamond Knife

Since its introduction by Fernandez-Moran, the diamond knife has gained wide spread usage as a common material for cutting of thin sections of biological and metallic materials into thin films for examination in the transmission electron microscope. With the development of high voltage E.M. and scanning transmission E.M., microtomy applications will become increasingly important in the preparation of specimens. For those who can afford it, the diamond knife will thus continue to be an important tool to accomplish this effort until a cheaper but equally strong and sharp tool is found to replace the diamond, glass not withstanding.In Figs. 1 thru 3, a first attempt was made to examine the edge of a used (β=45°) diamond knife by means of the scanning electron microscope. Because diamond is conductive, first examination was tried without any coating of the diamond. However, the contamination at the edge caused severe charging during imaging. Next, a thin layer of carbon was deposited but charging was still extensive at high magnification - high voltage settings. Finally, the knife was given a light coating of gold-palladium which eliminated the charging and allowed high magnification micrographs to be made with reasonable resolution.

Determination of the phase structure of polymerblends by electron microscopy

1978 ◽  
Vol 36 (1) ◽  
pp. 492-493
Author(s):  
Dr. G. Kaemof
Keyword(s):  
Screw Extruder ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Single Screw Extruder ◽  
Transmission Electron ◽  
The Matrix ◽  
Twin Screw ◽  
Special Case ◽  
Microscopic Investigations

A mixture of polycarbonate (PC) and styrene-acrylonitrile-copolymer (SAN) represents a very good example for the efficiency of electron microscopic investigations concerning the determination of optimum production procedures for high grade product properties.The following parameters have been varied:components of charge (PC : SAN 50 : 50, 60 : 40, 70 : 30), kind of compounding machine (single screw extruder, twin screw extruder, discontinuous kneader), mass-temperature (lowest and highest possible temperature).The transmission electron microscopic investigations (TEM) were carried out on ultra thin sections, the PC-phase of which was selectively etched by triethylamine.The phase transition (matrix to disperse phase) does not occur - as might be expected - at a PC to SAN ratio of 50 : 50, but at a ratio of 65 : 35. Our results show that the matrix is preferably formed by the components with the lower melting viscosity (in this special case SAN), even at concentrations of less than 50 %.

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