Confocal human cell

SmithPJ, BluntN, Wiltshire M, etal. Characteristics ofa novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy. Cytometry. 2000 40(4) 280-291. 

Results from Confocal Raman Microspectroscopy of Human Cells... 

As human skin fibroblasts are recalcitrant to transfection, we wished to include an easily traceable marker, which can either be used as an in-frame fusion protein, or as a separately expressed protein. The widely applicable green fluorescent protein (GFP) reporter system, originally isolated from the jellyfish Aequorea victoria, and adapted for efficient expression in human cells and for, elevated levels of fluorescence, provides these properties. The GFP system has been used for example, to study protein trafficking to the mitochondrial matrix and the mitochondrial outer membrane. Therefore, by using confocal laser scanning imaging we may get an impression of the intracellular locahzation of a CPTl-GFP fusion protein after transient or stable transfection. 

Our imaging studies have demonstrated that it is possible to image C60 and SWNTs in both fixed and live cells using STEM, TEM and confocal microscopy techniques. We exposed human macrophage cells to C60 and SWNTs and found that both C60... 

Figure 26. Reconstruction of the tunica intima on the inner surface of a clinically used polyethylene terephtalate vascular prosthesis. A non-modified inner surface of the prosthesis, B immobilization of defined assemblies of protein molecules (e.g., collagenfiarninin or collagen+fibrin) on the inner surface of the graft, C immunofluorescence of von Willebrand factor, a marker of the identity a differentiation of vascular endothelial cells, in human saphenous vein endothelial cells in cultures on the inner surface of a prosthesis coated with collagen and larninin, D detail of a layer of endothelial cells growing on a layer of collagen and fibrin. Note well developed talin-containing focal adhesion plaques. A, B conventional optical microscope, C, D confocal microscope Leica DM 2500 [30,31].

Figure 27. Human osteoblast-like MG 63 cells in cultures on porous (A) or fibrous (B) poly(L-lactide-co-glycolide) scaffolds. A A summarizing picture of horizontal optical sections. The depth of cell ingrowth into the pores (average pore diameter of 400-600 mm) is indicated by spectral colors (blue 0-60 mm, green 80-160 mm, yellow 180-220 mm, orange 240-300 mm, red 320-400 mm, violet 420-480 mm). Day 14 after seeding, cells stained with propidium iodide. B cells grown for 4 days in static culture followed by 2 days in dynamic perfusion cell culture system. Cell membrane stained with Texas Red C2-maleimide and the nuclei counterstained with Hoechst 33342. Leica TCS SP2 confocal microscope, objective 5x (A) or lOx (B) [37].

Variations on the filter-based assay have been designed to approximate more physiological contexts. Such assays include tumor cell invasion across a confluent cell monolayer (e.g., endothelial cells (EC) as a surrogate for intravasation or extravasation during hematogenous metastasis (24)) and ovarian carcinoma invasion of mesothelial cell monolayers (25). Additionally, 1 mm thick slices of human brain tissue have been used as a tissue barrier on Transwell filters with invasion of GFP-labeled glioma cells measured by confocal microscopy (26). 

Ocular damaging and irritant agents can be identified and evaluated by the Draize rabbit test [114]. However, more recently this test has been criticized on the basis of ethical considerations and unreliable prognosis of human response. Alternative methods such as the evaluation of toxicity on ocular cell cultures have been recommended and are being indicated as promising prognostic tools [115-120]. Direct confocal microscopic analysis [121], hydration level of isolated corneas [122], and various other tests on isolated corneas or animal eyes have also been proposed for evaluation of ocular toxic effects. 

Western blots of microsomal fractions from transfected HEK-293 cells with the PLN-L39stop mutant, indicated that the PLN-L39stop protein could not be detected. In addition, confocal microscopy in HEK-293 cells transfected with PLN-L39stop revealed detectable immunoreactive protein signals in a small percent of cells and the PLN-mutant was mainly localized to the cell membrane, compared with PLN-WT, which localized to the endoplasmic reticulum. Consistent with these findings, human PLN-L39stop homozygous ventricles had no detectable PLN. 

In this chapter, we provide protocols to determine the ability of a peptide to mediate DNA internalization in cultured human tumor cells. Fluorescence-assisted cell sorting (FACS) analysis is used to obtain quantitative data on the time and temperature dependence of macromolecular delivery. Confocal microscopy is used to study the subcellular localization in both fixed and live cells. Fluorescently labeled transferrin and dextran are used to label the clathrin-dependent (15) and the non-clathrin, non-caveolar (16) endocytic compartments, respectively. Expression of a caveolin-l-YFP fusion protein is used to label cell surface caveolae and intracellular caveosomes (17). Finally a protocol, for the overexpression of dominant-negative dynamin [GTPase deficient dynamin-2 containing the amino acid substitution K44A (18)] is provided to evaluate the dynamin dependence of the uptake mechanism. 

Figure 6.7. Transmission images of human neuroblastoma cells obtained with confocal microscopy showing the morphological alterations Induced by palytoxin (center) and ostreocIn-D (right) after 4 hours Incubation. Normal cells are at left.

Color Plate 1. Confocal imaging of human aortic smooth muscle cells transfected with an adenovims (Fig. 1, Chapter 4 see full caption and discussion on pp. 117-118). 

We developed adenoviral constructs of the human a1B-AR and the oqD-AR coupled with GFP. These constructs infected human aortic smooth muscle cells (Cascade Biologies, Portland, OR) with approx 70% efficiency (35). Confocal images of infected cells (Fig. 4 see Color Plate 1 following p. 148.) show that the a1B-AR/GFP is primarily expressed on the cell surface of vascular smooth muscle cells, although some cytoplasmic expression is also observed. This localization pattern agrees with the work of MacKenzie et al. (34). 

Fig. 4 Confocal laser scans of human hepatocyte spheroids, (a) Human hepatocyte spheroid. Nuclei appear blue (DAPI staining). Bile canaliculi are visualized by green fluorescence (staining of the bile canalicular marker DPPIV). (b) Similar spheroid as shown in (a), with inclusion of sinusoidal endothelial cells (red) into the hanging drop culture. The endothelial cells do not form vessel-like structures as in vivo but build an epithelium at the surface of the spheroid, (c) Kupffer cells integrated into a spheroid of hepatocytes. (d) Reconstructed bile canalicular network of a spheroid of human hepatocytes. (e) Bile canalicular network of liver tissue...

Oddi S, Bari M, Battista N, Barsacchi D, Cozzani I, Maccarrone M (2005) Confocal microscopy and biochemical analysis reveal spatial and functional separation between anandamide uptake and hydrolysis in human keratinocytes. Cell Mol Life Sci 62 386-395... 

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